Processes for producing ethanol

ABSTRACT

The present invention relates to processes for producing fermentation products from starch-containing material, wherein a thermostable alpha-amylase and optionally a thermostable protease are present and/or added during liquefaction. The invention also relates to a composition suitable for use in a process of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/407,840 filed May 9, 2019, now allowed, which is a continuation ofU.S. application Ser. No. 15/727,952 filed Oct. 9, 2017, now allowed,which is a continuation of U.S. application Ser. No. 13/994,310 filedOct. 1, 2013, now U.S. Pat. No. 9,816,112, which is a 35 U.S.C. 371national application of PCT/US2011/066559 filed Dec. 21, 2011 whichclaims priority or the benefit under 35 U.S.C. 119 of U.S. provisionalapplication Nos. 61/426,039 and 61/566,373 filed Dec. 22, 2010 and Dec.2, 2011, respectively, the contents of which are fully incorporatedherein by reference.

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentationproducts from starch-containing material. The invention also relates toa composition suitable for use in a process of the invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Production of fermentation products, such as ethanol, fromstarch-containing material is well-known in the art. Industrially twodifferent kinds of processes are used today. The most commonly usedprocess, often referred to as a “conventional process”, includingliquefying gelatinized starch at high temperature using typically abacterial alpha-amylase, followed by simultaneous saccharification andfermentation carried out in the presence of a glucoamylase and afermentation organism. Another well known process, often referred to asa “raw starch hydrolysis”-process (RSH process) includes simultaneouslysaccharifying and fermenting granular starch below the initialgelatinization temperature typically in the presence of an acid fungalalpha-amylase and a glucoamylase.

Despite significant improvement of fermentation product productionprocesses over the past decade a significant amount of residual starchmaterial is not converted into the desired fermentation product, such asethanol. At least some of the unconverted residual starch material,e.g., sugars and dextrins, is in the form of non-fermentable Maillardproducts.

Therefore, there is still a desire and need for providing processes forproducing fermentation products, such as ethanol, from starch-containingmaterial that can provide a higher fermentation product yield comparedto a conventional process.

SUMMARY OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as ethanol from starch-containing material using afermenting organism. The invention also relates to a compositionsuitable for use in a process of the invention.

In the first aspect the invention relates to processes for producingfermentation products, such as ethanol, from starch-containing materialcomprising the steps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

In an embodiment a carbohydrate-source generating enzyme, in particulara thermostable glucoamylase, and/or a pullulanase is(are) present and/oradded during liquefaction in step i).

In a second aspect the invention relates to compositions comprising analpha-amylase and a protease, wherein

i) the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) ofat least 10; and

ii) the protease has a thermostability value of more than 20% determinedas Relative Activity at 80° C./70° C.

In an embodiment the composition further comprises a carbohydrate-sourcegenerating enzyme, in particular a thermostable glucoamylase, and/or apullulanase.

In an embodiment a second alpha-amylase is present and/or added duringliquefaction step i).

In an embodiment the invention relates to a composition comprising:

i) an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂)of at least 10 derived from Bacillus stearothermophilus;

ii) a protease having a thermostability value of more than 20%determined as Relative Activity at 80° C./70° C. derived derived fromPyrococcus furiosus and/or Thermoascus aurantiacus; and optionally

iii) a glucoamylase derived from Penicillium oxalicum.

In an embodiment the composition comprises a second alpha-amylase.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a comparison of 54 hour ethanol for liquefactions (85° C.)prepared with Alpha-Amylase 1407 with and without Protease Pfu orGlucoamylase PE001 at pH 4.8.

FIG. 2 shows the peak and break viscosity at 32% DS for the experimentin Example 10 comparing

Alpha-Amylase A (1.4 micro g) (pH 5.8);

Alpha-Amylase 1407 (1.4 micro g)+Glucoamylase PE001 (10 microg)+Protease 196 (1 micro g) (pH 4.8);

Alpha-Amylase A (0.35 micro g)+Alpha-Amylase 1407 (1.4 microg)+Glucoamylase PE001 (10 micro g)+Protease 196 (1 micro g) (pH 4.8);

Alpha-Amylase A (0.7 micro g)+Alpha-Amylase 1407 (1.4 microg)+Glucoamylase PE001 (10 micro g)+Protease 196 (1 micro g) (pH 4.8);

FIG. 3 shows the final viscosity at 32% DS at 32° C. for the experimentin Example 10 comparing

Alpha-Amylase A (1.4 micro g) (pH 5.8);

Alpha-Amylase 1407 (1.4 micro g)+Glucoamylase PE001 (10 microg)+Protease 196 (1 micro g) (pH 4.8);

Alpha-Amylase A (0.35 micro g)+Alpha-Amylase 1407 (1.4 microg)+Glucoamylase PE001 (10 micro g)+Protease 196 (1 micro g) (pH 4.8);

Alpha-Amylase A (0.7 micro g)+Alpha-Amylase 1407 (1.4 microg)+Glucoamylase PE001 (10 micro g)+Protease 196 (1 micro g) (pH 4.8).

FIG. 4 shows the peak and break viscosity at 32% DS for the experimentin Example 10 comparing Alpha-Amylase A (1.4 micro g) (pH 5.8) andAlpha-Amylase A (1.4 micro g)+Glucoamylase PE001 (10 micro g)+Protease196 (1 micro g) (pH 4.8).

FIG. 5 shows the final viscosity at 32% DS, at 32° C. for the experimentin Example 10 comparing Alpha-Amylase A (1.4 micro g) (pH 5.8) andAlpha-Amylase A (1.4 micro g)+Glucoamylase PE001 (10 micro g)+Protease196 (1 micro g) (pH 4.8).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to processes of producing fermentationproducts, such as ethanol from starch-containing material using afermenting organism. The invention also relates to a compositionsuitable for use in a process of the invention.

The inventors have shown that a process of the invention has a number ofadvantages. As shown in the Examples a process of the invention resultsin a higher ethanol yield. Other benefits, includes a reduced need forusing H₂SO₄ for pH adjustment. This results in less sulfur downstream inthe DDGS, less front-end fouling, less beerstone, and less phytateprecipitation.

A process of the invention also results in reduced loss of sugars anddextrins to Maillard products. The DDGS color is improved and the heatexchanger lifetime (less solids) is extended. Furthermore, due to thehigher thermostability of the enzymes used the enzyme dose may bereduced. A process of the invention requires limited changes to existingprocess and process equipment and thus limited capital investment.

By having a thermostable alpha-amylase and a second alpha-amylase asdefined herein in liquefaction the peak viscosity, e.g., in slurry tankis (further) reduced. This result in less energy spent for mixing. Alsohaving a lower average viscosity improves the mixing of the mash/starchin the slurry tank and its pumping through the liquefaction process.

In the first aspect the invention relates to processes for producingfermentation products from starch-containing material comprising thesteps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

In a preferred embodiment step ii) and iii) are carried out eithersequentially or simultaneously. The thermostable alpha-amylase andoptionally a thermostable protease and optionally thecarbohydrate-source generating enzyme, preferably thermostableglucoamylase, and/or optionally a pullulanase may be added before and/orduring liquefaction step i). Examples of thermostable alpha-amylases canbe found in the “Alpha-Amylase Present and/or Added During Liquefaction”section below. Examples of thermostable proteases can be found in the“Protease Present and/or Added During Liquefaction”-section below. Acomposition of the invention may suitably be used in a process of theinvention. However, the enzyme components may also be added separately.

In a preferred embodiment the pH during liquefaction is between 4.5-4.8.

In an embodiment a carbohydrate-source generating enzyme is also presentduring liquefaction. In a preferred embodiment the carbohydrate-sourcegenerating enzymes is a thermostable glucoamylase. In an embodiment thecarbohydrate-source generating enzyme is different from the one usedduring saccharification in step ii) and/or fermentation in step iii).

Examples of “carbohydrate-source generating enzymes”, including inparticular glucoamylases, can be found in the “Carbohydrate-SourceGenerating Enzyme Present and/or Added During Liquefaction”-sectionbelow. Examples of thermostable glucoamylases can be found in the“Glucoamylase Present and/or Added During Liquefaction”-section below.

In an embodiment, the process of the invention further comprises, priorto the step i), the steps of:

a) reducing the particle size of the starch-containing material,preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

The starch-containing starting material, such as whole grains, may bereduced in particle size, e.g., by milling, in order to open up thestructure and allowing for further processing. Generally there are twotypes of processes: wet and dry milling. In dry milling whole kernelsare milled and used. Wet milling gives a good separation of germ andmeal (starch granules and protein) and is often applied at locationswhere the starch hydrolysate is used in production of, e.g., syrups.Both dry and wet milling is well known in the art of starch processing.According to the invention dry milling is preferred. In an embodimentthe particle size is reduced to between 0.05 to 3.0 mm, preferably0.1-0.5 mm, or so that at least 30%, preferably at least 50%, morepreferably at least 70%, even more preferably at least 90% of thestarch-containing material fit through a sieve with a 0.05 to 3.0 mmscreen, preferably 0.1-0.5 mm screen. In another embodiment at least50%, preferably at least 70%, more preferably at least 80%, especiallyat least 90% of the starch-containing material fit through a sieve with#6 screen.

The aqueous slurry may contain from 10-55 w/w-% dry solids (DS),preferably 25-45 w/w-% dry solids (DS), more preferably 30-40 w/w-% drysolids (DS) of starch-containing material. The slurry is heated to abovethe gelatinization temperature, preferably for between 80-90° C., pH4.5-4.8 for around 15-60 minutes.

The thermostable alpha-amylase, optional thermostable protease andoptional carbohydrate-source generating enzyme, in particularthermostable glucoamylase, and/or optional pullulanase may be added tothe aqueous slurry to initiate liquefaction (thinning). In an embodimentonly a part of the enzyme blend (composition of the invention) is addedto the aqueous slurry, while the rest of the enzyme is added duringliquefaction step i). Liquefaction step i) is typically carried out at80-90° C., pH 4.5-4.8 for 1-3 hours.

The aqueous slurry may in an embodiment be jet-cooked to furthergelatinize the slurry before being subjected to liquefaction in step i).The jet-cooking may be carried out at a temperature between 110-145° C.,preferably 120-140° C., such as 125-135° C., preferably around 130° C.for about 1-15 minutes, preferably for about 3-10 minutes, especiallyaround about 5 minutes.

Saccharification and Fermentation

One or more carbohydrate-source generating enzymes, in particularglucoamylases, are present and/or added during saccharification step ii)and/or fermentation step iii). The carbohydrate-source generating enzymemay preferably be a glucoamylase, but may also be an enzyme selectedfrom the group consisting of: beta-amylase, maltogenic amylase andalpha-glucosidase.

Examples of carbohydrate-source generating enzyme, includingglucoamylases, can be found in the “Carbohydrate-Source GeneratingEnzyme Present and/or Added During Saccharification and/orFermentation”-section below.

When doing sequential saccharification and fermentation thesaccharification step ii) may be carried out using conditions well-knownin the art. For instance, the saccharification step ii) may last up tofrom about 24 to about 72 hours, however, it is common to do only apre-saccharification of typically 40-90 minutes at a temperature between30-65° C., typically about 60° C., followed by saccharification duringfermentation in simultaneous saccharification and fermentation (“SSF).Saccharification is typically carried out at temperatures from 20-75°C., preferably from 40-70° C., typically around 60° C., and at a pHbetween 4 and 5, normally at about pH 4.5.

Simultaneous saccharification and fermentation (“SSF”) is widely used inindustrial scale fermentation product production processes, especiallyethanol production processes. When doing SSF the saccharification stepii) and the fermentation step iii) are carried out simultaneously. Thereis no holding stage for the saccharification, meaning that a fermentingorganism, such as yeast, and enzyme(s), may be added together. SSF areaccording to the invention typically carried out at a temperature from25° C. to 40° C., such as from 28° C. to 35° C., such as from 30° C. to34° C., preferably around about 32° C. In an embodiment fermentation isongoing for 6 to 120 hours, in particular 24 to 96 hours. In anembodiment the pH is between 3.5-5, in particular between 3.8 and 4.3.

Fermentation Medium

“Fermentation media” or “fermentation medium” which refers to theenvironment in which fermentation is carried out and which includes thefermentation substrate, that is, the carbohydrate source that ismetabolized by the fermenting organism. The fermentation medium maycomprise nutrients and growth stimulator(s) for the fermentingorganism(s). Nutrient and growth stimulators are widely used in the artof fermentation and include nitrogen sources, such as ammonia; urea,vitamins and minerals, or combinations thereof.

Fermenting Organisms

The term “Fermenting organism” refers to any organism, includingbacterial and fungal organisms, suitable for use in a fermentationprocess and capable of producing the desired fermentation product.Especially suitable fermenting organisms are able to ferment, i.e.,convert, sugars, such as glucose or maltose, directly or indirectly intothe desired fermentation product, such as ethanol. Examples offermenting organisms include fungal organisms, such as yeast. Preferredyeast includes strains of Saccharomyces spp., in particular,Saccharomyces cerevisiae.

In one embodiment the fermenting organism is added to the fermentationmedium so that the viable fermenting organism, such as yeast, count permL of fermentation medium is in the range from 10⁵ to 10¹², preferablyfrom 10⁷ to 10¹⁰, especially about 5×10⁷.

Commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™yeast (available from Fermentis/Lesaffre, USA), FALI (available fromFleischmann's Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR(available from NABC—North American Bioproducts Corporation, GA, USA),GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL(available from DSM Specialties).

Starch-Containing Materials

Any suitable starch-containing material may be used according to thepresent invention. The starting material is generally selected based onthe desired fermentation product. Examples of starch-containingmaterials, suitable for use in a process of the invention, include wholegrains, corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum,rice, peas, beans, or sweet potatoes, or mixtures thereof or starchesderived there from, or cereals. Contemplated are also waxy and non-waxytypes of corn and barley.

Fermentation Products

The term “fermentation product” means a product produced by a processincluding a fermentation step using a fermenting organism. Fermentationproducts contemplated according to the invention include alcohols (e.g.,ethanol, methanol, butanol); organic acids (e.g., citric acid, aceticacid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones(e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H₂ andCO₂); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins(e.g., riboflavin, B₁₂, beta-carotene); and hormones. In a preferredembodiment the fermentation product is ethanol, e.g., fuel ethanol;drinking ethanol, i.e., potable neutral spirits; or industrial ethanolor products used in the consumable alcohol industry (e.g., beer andwine), dairy industry (e.g., fermented dairy products), leather industryand tobacco industry. Preferred beer types comprise ales, stouts,porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer,low-alcohol beer, low-calorie beer or light beer. Preferred fermentationprocesses used include alcohol fermentation processes. The fermentationproduct, such as ethanol, obtained according to the invention, maypreferably be used as fuel, that typically is blended with gasoline.However, in the case of ethanol it may also be used as potable ethanol.

Recovery

Subsequent to fermentation the fermentation product may be separatedfrom the fermentation medium. The slurry may be distilled to extract thedesired fermentation product. Alternatively the desired fermentationproduct may be extracted from the fermentation medium by micro ormembrane filtration techniques. The fermentation product may also berecovered by stripping or other method well known in the art.

Alpha-Amylase Present and/or Added During Liquefaction

According to the invention a thermostable alpha-amylase is presentand/or added during liquefaction optionally together with a thermostableprotease, and optionally a carbohydrate-source generating enzyme,preferably a thermostable glucoamylase, and/or optionally a pullulanase.According to the invention the alpha-amylase has high activity towardstarch solubilisation in liquefaction at pH 4.5 to 5.0 and highthermostability at pH 4.5-5.0 and 80-90° C., preferably 4.5-4.8, around85° C.

More specifically the alpha-amylase used in a process of the inventionhas a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10determined as described in Example 1.

In a preferred embodiment T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂, isat least 15, such as at least 20, such as at least 25, such as at least30, such as at least 40, such as at least 50, such as at least 60, suchas between 10-70, such as between 15-70, such as between 20-70, such asbetween 25-70, such as between 30-70, such as between 40-70, such asbetween 50-70, such as between 60-70.

In an embodiment the thermostable alpha-amylase is a Bacillusstearothermophilus alpha-amylase variant having at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID NO: 1herein.

In an embodiment of the invention the alpha-amylase is an bacterialalpha-amylase, preferably derived from the genus Bacillus, especially astrain of Bacillus stearothermophilus, in particular the Bacillusstearothermophilus as disclosed in WO 99/019467 as SEQ ID NO: 3 or SEQID NO: 1 herein with the double deletion I181+G182 and substitutionN193F, further comprising the following mutations:

-   -   V59A+Q89R+G112D+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+D269E+D281N;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+I270L;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+H274K;    -   V59A+Q89R+E129V+K177L+R179E+K220P+N224L+Q254S+Y276F;    -   V59A+E129V+R157Y+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+H208Y+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+H274K;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+D281N;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   V59A+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+G416V;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S;    -   V59A+E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   A91L+M96I+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+Y276F+L427M;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+M284T;    -   E129V+K177L+R179E+K220P+N224L+S242Q+Q254S+N376*+I377*;    -   E129V+K177L+R179E+K220P+N224L+Q254S;    -   E129V+K177L+R179E+K220P+N224L+Q254S+M284T;    -   E129V+K177L+R179E+S242Q;    -   E129V+K177L+R179V+K220P+N224L+S242Q+Q254S;    -   K220P+N224L+S242Q+Q254S;    -   M284V.

The thermostable alpha-amylase may be a truncated Bacillusstearothermophilus alpha-amylase, preferably to have around 491 aminoacids.

Second Alpha-Amylase Present and/or Added During Liquefaction

When a second alpha-amylase is present and/or added during liquefactionstep i) a positive viscosity reducing effect is obtained. As can be seenfrom Example 10 the combination of a thermostable alpha-amylase (e.g.,Alpha-Amylase BE1407) with or without the presence of a thermostableprotease (e.g., Protease 196) and thermostable glucoamylase (e.g.,Glucoamylase PO) and further a second alpha-amylase (e.g. Alpha-amylaseA) results in decrease peak viscosity and final viscosity.

Therefore, in this aspect of the invention a second alpha-amylase isadded during liquefaction step i). The second alpha-amylase may be lessthermostable and/or less efficient at pH 4.5, 85° C., 0.12 mM CaCl₂, oraround pH 4.8, than a thermostable alpha-amylase defined herein addedand/or present during liquefaction according to the invention.

In an embodiment the second alpha-amylase is of bacterial origin.

In an embodiment the second alpha-amylase is derived from a strain ofthe genus Bacillus, such as a strain of Bacillus stearothermophilus, inparticular a variant of a Bacillus stearothermophilus alpha-amylase,such as the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1herein. The second alpha-amylase may be a truncated Bacillusstearothermophilus alpha-amylase, preferably to have around 491 aminoacids.

In an embodiment second alpha-amylase has at least 80%, more preferablyat least 85%, more preferably at least 90%, more preferably at least91%, more preferably at least 92%, even more preferably at least 93%,most preferably at least 94%, and even most preferably at least 95%,such as even at least 96%, at least 97%, at least 98%, at least 99%, orat least 100% identity to the mature part of the polypeptide of SEQ IDNO: 3 disclosed in WO 99/019467 or SEQ ID NO: 1 herein.

In an embodiment the second alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂) of below 10 determined as described in Example 1.

In an embodiment the second alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂) of below 8, such as below 7, such as below 6, such asbelow 5.

In an embodiment the second alpha-amylase has a T½ (min) at pH 4.5, 85°C., 0.12 mM CaCl₂) between 2 and 10, such as between 3 and 8, such asabove 4 to 10, such as above 4 to 8.

In an embodiment the second alpha-amylase may be derived from Bacillusstearothermophilus and may have the following mutations: I181*+G182* orI181*+G182*+N193F (using SEQ ID NO: 1 for numbering).

In an embodiment the invention relates to a process for producingfermentation products from starch-containing material comprising thesteps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10, and further a second alpha-amylase having        a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of less than 10;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C.;

ii) saccharifying using a glucoamylase;

iii) fermenting using a fermenting organism.

Protease Present and/or Added During Liquefaction

According to the invention a thermostable protease may be present and/oradded during liquefaction together with a thermostable alpha-amylase,and optionally a carbohydrate-source generating enzyme, preferably athermostable glucoamylase, and/or optionally a pullulanase.

A protease used in a process of the invention has either

i) a thermostability value of more than 20% determined as RelativeActivity at 80° C./70° C.; and/or

ii) a thermostability value of more than 10% determined as RelativeActivity at 85° C./70° C.

In an embodiment the protease has a thermostability value:

-   -   of more than 30%, more than 40%, more than 50%, more than 60%,        more than 70%, more than 80%, more than 90% determined as        Relative Activity at 80° C./70° C., and/or    -   of more than 12%, more than 14%, more than 16%, more than 18%,        more than 20%, more than 25% determined as Relative Activity at        85° C./70° C.; and/or    -   of more that 20%, more than 30%, more than 40%, more than 50%,        more than 60%, more than 70%, more than 80%, more than 90%        determined as Remaining Activity at 80° C.; and/or    -   of more that 20%, more than 30%, more than 40%, more than 50%,        more than 60%, more than 70%, more than 80%, more than 90%        determined as Remaining Activity at 84° C. and/or.

Purified variants may have a themostability for above 90, above 100 at85° C. as determined using the Zein-BCA assay as disclosed in Example 3.

Determination of “Relative Activity” and “Remaining Activity” isdetermined as described in Example 2.

Proteases are classified on the basis of their catalytic mechanism intothe following groups: Serine proteases (S), Cysteine proteases (C),Aspartic proteases (A), Metallo proteases (M), and Unknown, or as yetunclassified, proteases (U), see Handbook of Proteolytic Enzymes, A. J.Barrett, N. D. Rawlings, J. F. Woessner (eds), Academic Press (1998), inparticular the general introduction part.

In a preferred embodiment the thermostable protease used in a process ofthe invention is a “metallo protease” defined as a protease belonging toEC 3.4.24 (metalloendopeptidases); preferably EC 3.4.24.39 (acid metalloproteinases).

To determine whether a given protease is a metallo protease or not,reference is made to the above “Handbook of Proteolytic Enzymes” and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Protease activity can be measured using any suitable assay, in which asubstrate is employed, that includes peptide bonds relevant for thespecificity of the protease in question. Assay-pH and assay-temperatureare likewise to be adapted to the protease in question. Examples ofassay-pH-values are pH 6, 7, 8, 9, 10, or 11. Examples ofassay-temperatures are 30, 35, 37, 40, 45, 50, 55, 60, 65, 70 or 80° C.

Examples of protease substrates are casein, such as Azurine-CrosslinkedCasein (AZCL-casein). Two protease assays are described below in the“Materials & Methods”-section, of which the so-called “AZCL-CaseinAssay” is the preferred assay.

In an embodiment the protease has at least 20%, such as at least 30%,such as at least 40%, such as at least 50%, such as at least 60%, suchas at least 70%, such as at least 80%, such as at least 90%, such as atleast 100% of the activity of the JTP196 protease variant or ProteasePfu determined by the AZCL-casein assay.

There are no limitations on the origin of the protease used in a processof the invention as long as it fulfills the thermostability propertiesdefined above. The protease may be a variant of, e.g., a wild-typeprotease as long as the protease has the thermostability propertiesdefined above. In a preferred embodiment the protease is a variant of ametallo protease as defined above. In an embodiment the protease used ina process of the invention is of fungal origin, such as a fungal metalloprotease, such as a fungal metallo protease derived from a strain of thegenus Thermoascus, preferably a strain of Thermoascus aurantiacus,especially Thermoascus aurantiacus CGMCC No. 0670 (classified as EC3.4.24.39).

In an embodiment the protease variant has at least 75% identitypreferably at least 80%, more preferably at least 85%, more preferablyat least 90%, more preferably at least 91%, more preferably at least92%, even more preferably at least 93%, most preferably at least 94%,and even most preferably at least 95%, such as even at least 96%, atleast 97%, at least 98%, at least 99%, but less than 100% identity tothe mature part of the polypeptide of SEQ ID NO: 2 disclosed in WO2003/048353 or the mature part of SEQ ID NO. 1 in WO 2010/008841 or SEQID NO: 3 herein.

In an embodiment the protease is a variant of the mature part of themetallo protease shown in SEQ ID NO: 2 disclosed in WO 2003/048353 orthe mature part of SEQ ID NO: 1 in WO 2010/008841 and shown as SEQ IDNO: 3 herein with the following mutations:

-   -   S5*+N26R+D79L+S87P+A112P+D142L;    -   S5*+D79L+S87P+A112P+D142L;    -   N26R+T46R+D79L+S87P+A112P+D142L;    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+D79L+Y82F+D104P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+T124V+D142L;    -   A27K+D79L+S87P+A112P+A126V+D142L;    -   A27K+D79L+S87P+A112P+D142L;    -   A27K+Y82F+S87G+D104P+A112P+A126V+D142L;    -   A27K+Y82F+D104P+A112P+A126V+D142L;    -   S36P+D79L+S87P+A112P+D142L;    -   A37P+D79L+S87P+A112P+D142L;    -   S38T+D79L+S87P+A112P+A126V+D142L;    -   T46R+D79L+S87P+T116V+D142L;    -   S49P+D79L+S87P+A112P+D142L;    -   S50P+D79L+S87P+A112P+D142L;    -   S70V+D79L+Y82F+S87G+Y97W+A112P+D142L;    -   S70V+D79L+Y82F+S87G+A112P+D142L;    -   D79L+P81R+S87P+A112P+D142L;    -   D79L+Y82F+S87G+Y97W+D104P+A112P+D142L;    -   D79L+Y82F+S87G+D104P+A112P+D142L;    -   D79L+Y82F+S87G+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   D79L+Y82F+S87P+A112P+T124V+D142L;    -   D79L+Y82F+S87P+A112P+A126V+D142L;    -   D79L+Y82F+S87P+A112P+D142L;    -   D79L+S87P+N98C+A112P+G135C+D142L;    -   D79L+S87P+D104P+A112P+D142L;    -   D79L+S87P+A112P+T124V+A126V+D142L;    -   D79L+S87P+A112P+T124V+D142L;    -   D79L+S87P+A112P+D142L;    -   D79L+S87P+A112P+D142L+T141C+M161C;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.

In an embodiment the thermostable protease present and/or added duringliquefaction step i) is derived from a strain of Pyrococcus, such as astrain of Pyrococcus furiosus. In an embodiment the protease is oneshown as SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-E1 (Takara ShuzoCompany). In another embodiment the protease is one disclosed in SEQ IDNO: 13 herein or a protease having at least 80% identity, such as atleast 85%, such as at least 90%, such as at least 95%, such as at least96%, such as at least 97%, such as at least 98%, such as at least 99%identity to SEQ ID NO: 1 in U.S. Pat. No. 6,358,726-E1 or SEQ ID NO: 13herein. The Pyrococcus furiosus protease can be purchased from TakaraShuzo Co. Ltd, Japan.

The Pyrococcus furiosus protease is a thermostable protease. Thecommercial product Pyrococcus furiosus protease (Pfu S) was found tohave a thermostability of 110% (80° C./70° C.) and 103% (90° C./70° C.)at pH 4.5 determined as described in Example 2 herein.

Carbohydrate-Source Generating Enzyme Present and/or Added DuringLiquefaction

According to the invention a carbohydrate-source generating enzyme,preferably a thermostable glucoamylase, is present and/or added duringliquefaction together with a thermostable alpha-amylase and optionally athermostable protease. As mentioned above a pullulanase may also bepresent and/or added during liquefaction step i).

The term “carbohydrate-source generating enzyme” includes any enzymesgenerating fermentable sugars. A carbohydrate-source generating enzymeis capable of producing a carbohydrate that can be used as anenergy-source by the fermenting organism(s) in question, for instance,when used in a process of the invention for producing a fermentationproduct, such as ethanol. The generated carbohydrates may be converteddirectly or indirectly to the desired fermentation product, preferablyethanol. According to the invention a mixture of carbohydrate-sourcegenerating enzymes may be used. Specific examples include glucoamylase(being glucose generators), beta-amylase and maltogenic amylase (beingmaltose generators).

In a preferred embodiment the carbohydrate-source generating enzyme is athermostable glucoamylase. The carbohydrate-source generating enzyme, inparticular thermostable glucoamylase, may be added together with orseparately from the thermostable alpha-amylase and optionally thethermostable protease.

In an embodiment the carbohydrate-source generating enzyme, preferably athermostable glucoamylase, has a Relative Activity heat stability at 85°C. of at least 20%, at least 30%, preferably at least 35%. In anembodiment the carbohydrate-generating enzyme is a glucoamylase having arelative activity at pH 4.5 of at least 80%, preferably at least 85%,preferably at least 90%, preferably at least 95%.

In a specific and preferred embodiment the carbohydrate-sourcegenerating enzyme is a thermostable glucoamylase, preferably of fungalorigin, preferably a filamentous fungi, such as from a strain of thegenus Penicillium, especially a strain of Penicillium oxalicum disclosedas SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802 (which ishereby incorporated by reference) and shown in SEQ ID NO: 9 or 14herein.

In a preferred embodiment the carbohydrate-source generating enzyme is avariant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in PCT/CN10/071753 published as WO 2011/127802 and shown in SEQ IDNOS: 9 and 14 herein, having a K79V substitution (using the maturesequence shown in SEQ ID NO: 14 for numbering). The K79V glucoamylasevariant has reduced sensitivity to protease degradation relative to theparent as disclosed in co-pending U.S. application No. 61/531,189 (whichis hereby incorporated by reference).

In a specific embodiment the carbohydrate-source generating enzyme is aglucoamylase, preferably derived from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed in PCT/CN10/071753published as WO 2011/127802. The glucoamylase may also be glucoamylasehaving at least 80%, more preferably at least 85%, more preferably atleast 90%, more preferably at least 91%, more preferably at least 92%,even more preferably at least 93%, most preferably at least 94%, andeven most preferably at least 95%, such as even at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the maturepolypeptide shown in SEQ ID NO: 2 in PCT/CN10/071753 published as WO2011/127802 and shown as SEQ ID NO: 9 and 14 herein.

Pullulanase Present and/or Added During Liquefaction

Optionally a pullulanase may be present and/or added during liquefactionstep i) together with a thermostable alpha-amylase and optionally athermostable protease. As mentioned above a carbohydrate-sourcegenerating enzyme, preferably a thermostable glucoamylase, may also bepresent and/or added during liquefaction step i).

The pullulanase may be present and/or added during liquefaction step i)and/or saccharification step ii) or simultaneous saccharification andfermentation.

Pullulanases (E.C. 3.2.1.41, pullulan 6-glucano-hydrolase), aredebranching enzymes characterized by their ability to hydrolyze thealpha-1,6-glycosidic bonds in, for example, amylopectin and pullulan.

Contemplated pullulanases according to the present invention include thepullulanases from Bacillus amyloderamificans disclosed in U.S. Pat. No.4,560,651 (hereby incorporated by reference), the pullulanase disclosedas SEQ ID NO: 2 in WO 01/151620 (hereby incorporated by reference), theBacillus deramificans disclosed as SEQ ID NO: 4 in WO 01/151620 (herebyincorporated by reference), and the pullulanase from Bacillusacidopullulyticus disclosed as SEQ ID NO: 6 in WO 01/151620 (herebyincorporated by reference) and also described in FEMS Mic. Let. 115:97-106 (1994).

Additional pullulanases contemplated according to the present inventionincluded the pullulanases from Pyrococcus woesei, specifically fromPyrococcus woesei DSM No. 3773 disclosed in WO 92/02614, and the matureprotein sequence disclosed as SEQ ID No: 6 herein.

In an embodiment the pullulanase is a family GH57 pullulanase. In anembodiment the pullulanase includes an X47 domain as disclosed in U.S.61/289,040 published as WO 2011/087836 (which are hereby incorporated byreference). More specifically the pullulanase may be derived from astrain of the genus Thermococcus, including Thermococcus litoralis andThermococcus hydrothermalis, such as the Thermococcus hydrothermalispullulanase shown in SEQ ID NO: 11 truncated at site X4 right after theX47 domain (i.e., amino acids 1-782 in SEQ ID NOS: 11 and 12 herein).The pullulanase may also be a hybrid of the Thermococcus litoralis andThermococcus hydrothermalis pullulanases or a T. hydrothermalis/T.litoralis hybrid enzyme with truncation site X4 disclosed in U.S.61/289,040 published as WO 2011/087836 (which is hereby incorporated byreference) and disclosed in SEQ ID NO: 12.

The pullulanase may according to the invention be added in an effectiveamount which include the preferred amount of about 0.0001-10 mg enzymeprotein per gram DS, preferably 0.0001-0.10 mg enzyme protein per gramDS, more preferably 0.0001-0.010 mg enzyme protein per gram DS.Pullulanase activity may be determined as NPUN. An Assay fordetermination of NPUN is described in the “Materials & Methods”-sectionbelow.

Suitable commercially available pullulanase products include PROMOZYMED, PROMOZYME™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int.,USA), and AMANO 8 (Amano, Japan).

Carbohydrate-Source Generating Enzyme Present and/or Added DuringSaccharification and/or Fermentation

According to the invention a carbohydrate-source generating enzyme,preferably a glucoamylase, is present and/or added duringsaccharification and/or fermentation.

In a preferred embodiment the carbohydrate-source generating enzyme is aglucoamylase, of fungal origin, preferably from a strain of Aspergillus,preferably A. niger, A. awamori, or A. oryzae; or a strain ofTrichoderma, preferably T. reesei; or a strain of Talaromyces,preferably T. emersonii.

Glucoamylase

According to the invention the glucoamylase present and/or added duringsaccharification and/or fermentation may be derived from any suitablesource, e.g., derived from a microorganism or a plant. Preferredglucoamylases are of fungal or bacterial origin, selected from the groupconsisting of Aspergillus glucoamylases, in particular Aspergillus nigerG1 or G2 glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), orvariants thereof, such as those disclosed in WO 92/00381, WO 00/04136and WO 01/04273 (from Novozymes, Denmark); the A. awamori glucoamylasedisclosed in WO 84/02921, Aspergillus oryzae glucoamylase (Agric. Biol.Chem. 55(4): 941-949 (1991)), or variants or fragments thereof. OtherAspergillus glucoamylase variants include variants with enhanced thermalstability: G137A and G139A (Chen et al., 1996, Prot. Eng. 9: 499-505);D257E and D293E/Q (Chen et al., 1995, Prot. Eng. 8: 575-582); N182 (Chenet al., 1994, Biochem. J. 301: 275-281); disulphide bonds, A246C(Fierobe et al., 1996, Biochemistry 35: 8698-8704; and introduction ofPro residues in position A435 and S436 (Li et al., 1997, Protein Eng.10: 1199-1204.

Other glucoamylases include Athelia rolfsii (previously denotedCorticium rolfsii) glucoamylase (see U.S. Pat. No. 4,727,026 and(Nagasaka et al., 1998, “Purification and properties of theraw-starch-degrading glucoamylases from Corticium rolfsii, Appl.Microbiol. Biotechnol. 50:323-330), Talaromyces glucoamylases, inparticular derived from Talaromyces emersonii (WO 99/28448), Talaromycesleycettanus (U.S. Pat. No. Re. 32,153), Talaromyces duponti, Talaromycesthermophilus (U.S. Pat. No. 4,587,215). In a preferred embodiment theglucoamylase used during saccharification and/or fermentation is theTalaromyces emersonii glucoamylase disclosed in WO 99/28448.

Bacterial glucoamylases contemplated include glucoamylases from thegenus Clostridium, in particular C. thermoamylolyticum (EP 135,138), andC. thermohydrosulfuricum (WO 86/01831) and Trametes cingulata,Pachykytospora papyracea; and Leucopaxillus giganteus all disclosed inWO 2006/069289; or Peniophora rufomarginata disclosed in WO 2007/124285;or a mixture thereof. Also hybrid glucoamylase are contemplatedaccording to the invention. Examples the hybrid glucoamylases disclosedin WO 2005/045018. Specific examples include the hybrid glucoamylasedisclosed in Tables 1 and 4 of Example 1 (which hybrids are herebyincorporated by reference).

In an embodiment the glucoamylase is derived from a strain of the genusPycnoporus, in particular a strain of Pycnoporus as described in U.S.61/264,977 published as WO 2011/066576 (SEQ ID NO: 2, 4 or 6), or from astrain of the genus Gloephyllum, in particular a strain of Gloephyllumas described in U.S. 61/406,741 published as WO 2011/068803 (SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or 16) or a strain of the genus Nigrofomes, inparticular a strain of Nigrofomes sp. disclosed in U.S. 61/411,044 orPCT/US10/058375 (SEQ ID NO: 2) (all references hereby incorporated byreference). Contemplated are also glucoamylases which exhibit a highidentity to any of above mention glucoamylases, i.e., at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or even 100%identity to any one of the mature parts of the enzyme sequencesmentioned above.

Contemplated are also glucoamylases which exhibit a high identity to anyof above mention glucoamylases, i.e., at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or even 100% identity to themature enzymes sequences mentioned above.

Glucoamylases may in an embodiment be added in an amount of 0.0001-20AGU/g DS, preferably 0.001-10 AGU/g DS, especially between 0.01-5 AGU/gDS, such as 0.1-2 AGU/g DS.

Commercially available compositions comprising glucoamylase include AMG200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ ECXEL and AMG™ E(from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from Genencor Int.);AMIGASE™ and AMIGASE™ PLUS (from DSM); G-ZYME™ G900, G-ZYME™ and G990 ZR(from Genencor Int.).

Maltogenic Amylase

The carbohydrate-source generating enzyme present and/or added duringsaccharification and/or fermentation may also be a maltogenicalpha-amylase. A “maltogenic alpha-amylase” (glucan1,4-alpha-maltohydrolase, E.C. 3.2.1.133) is able to hydrolyze amyloseand amylopectin to maltose in the alpha-configuration. A maltogenicamylase from Bacillus stearothermophilus strain NCIB 11837 iscommercially available from Novozymes A/S. Maltogenic alpha-amylases aredescribed in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628, whichare hereby incorporated by reference. The maltogenic amylase may in apreferred embodiment be added in an amount of 0.05-5 mg totalprotein/gram DS or 0.05-5 MANU/g DS.

A Composition Comprising Alpha-Amylase and Protease

A composition of the invention comprises a thermostable alpha-amylaseand a thermostable protease. The composition may also optionallycomprise a thermostable carbohydrate-source generating enzyme andoptionally a pullulanase.

Therefore, in this aspect the invention relates to compositioncomprising an alpha-amylase and a protease, wherein the

i) alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of atleast 10;

ii) protease having a thermostability value of more than 20% determinedas Relative Activity at 80° C./70° C.

The composition optionally further comprises a carbohydrate-sourcegenerating enzyme. Said carbohydrate-source generating enzyme may be athermostable glucoamylase having a Relative Activity heat stability at85° C. of at least 20%, at least 30%, preferably at least 35%.

The thermostable alpha-amylase is preferably a bacterial alpha-amylase,in particular of the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as a variant of one shown in SEQID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein. Alpha-amylase variantsare described further in the “Alpha-Amylase Present and/or Added DuringLiquefaction”-section above. The alpha-amylase may have a T½ (min) at pH4.5, 85° C., 0.12 mM CaCl₂) of at least 15, such as at least 20, such asat least 25, such as at least 30, such as at least 40, such as at least50, such as at least 60, such as between 10-70, such as between 15-70,such as between 20-70, such as between 25-70, such as between 30-70,such as between 40-70, such as between 50-70, such as between 60-70.

In an embodiment the alpha-amylase is selected from the group ofBacillus stearomthermphilus alpha-amylase variants, in particulartruncated to be 491 amino acids long, with mutations selected from thegroup of:

-   -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+E129V+K177L+R179E; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S        (using SEQ ID NO: 1 herein for numbering).

It should be understood that these alpha-amylases are only specificexamples. Any alpha-amylase disclosed above in the “Alpha-AmylasePresent and/or Added During Liquefaction”-section above may be used asthe alpha-amylase component in a composition of the invention.

The protease has a thermostability of:

i) more than 30%, more than 40%, more than 50%, more than 60%, more than70%, more than 80%, more than 90% determined as Relative Activity at 80°C./70° C.; or

ii) more than 12%, more than 14%, more than 16%, more than 18%, morethan 20%, more than 25% determined as Relative Activity at 85° C./70° C.

In an embodiment the protease is a variant of the metallo protease showin SEQ ID NO: 3 derived from Thermoascus aurantiacus CGMCC No. 0670.

In a specific preferred embodiment the protease is a variant of themetallo protease derived from Thermoascus aurantiacus disclosed as themature part of SEQ ID NO. 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein withmutations selected from the group of:

-   -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+S87P+A112P+D142L; and    -   D79L+S87P+D142L.

In another preferred embodiment the protease is derived from a strain ofPyrococcus furiosus, such as the one shown in SEQ ID NO: 1 in U.S. Pat.No. 6,258,726 or SEQ ID NO: 13 herein.

In another embodiment the protease is one disclosed in SEQ ID NO: 13herein or a protease having at least 80% identity, such as at least 85%,such as at least 90%, such as at least 95%, such as at least 96%, suchas at least 97%, such as at least 98%, such as at least 99% identity toSEQ ID NO: 1 in U.S. Pat. No. 6,358,726-B1 or SEQ ID NO: 13 herein. ThePyroccus furiosus protease can be purchased from Takara Shuzo Co. Ltd,Japan.

The Pyrococcus furiosus protease is a thermostable protease. Thecommercial product Pyrococcus furiosus protease (Pfu S) was found tohave a thermostability of 110% (80° C./70° C.) and 103% (90° C./70° C.)at pH 4.5 determined as described in Example 2 herein.

It should be understood that these proteases are only examples. Anyprotease disclosed above in the “Protease Present and/or Added DuringLiquefaction” section above may be used as the protease component in acomposition of the invention.

A composition of the invention may optionally further comprise acarbohydrate-source generating enzyme, in particular a glucoamylase,which has a heat stability at 85° C., pH 5.3, of at least 30%,preferably at least 35%.

In a preferred embodiment the carbohydrate-source generating enzyme is aglucoamylase having a relative activity of at least 80%, preferably atleast 85%, preferably at least 90% at pH 4.5.

In a preferred embodiment carbohydrate-source generating enzyme is aglucoamylase having a pH stability at pH 4.5 of at least at least 80%,at least 85%, at least 90%, at least 95%, at least 100%.

Determination heat stability, and pH stability is described in theExample 4.

In a specific embodiment the carbohydrate-source generating enzyme is aglucoamylase, preferably derived from a strain of the genus Penicillium,especially a strain of Penicillium oxalicum disclosed in PCT/CN10/071753published as WO 2011/127802. The glucoamylase may also be glucoamylasehaving at least 80%, more preferably at least 85%, more preferably atleast 90%, more preferably at least 91%, more preferably at least 92%,even more preferably at least 93%, most preferably at least 94%, andeven most preferably at least 95%, such as even at least 96%, at least97%, at least 98%, at least 99% or 100% identity to the maturepolypeptide shown in SEQ ID NO: 2 in PCT/CN10/071753 published as WO2011/127802.

In a preferred embodiment the carbohydrate-source generating enzyme is avariant of the Penicillium oxalicum glucoamylase disclosed as SEQ ID NO:2 in PCT/CN10/071753 published as WO 2011/127802 and shown in SEQ ID NO:9 and 14 herein, having a K79V substitution (using the mature sequenceshown in SEQ ID NO: 14 for numbering). The K79V glucoamylase variant hasreduced sensitivity to protease degradation relative to the parent asdisclosed in co-pending U.S. application No. 61/531,189 (which is herebyincorporated by reference).

A composition of the invention may further comprise a pullulanase. In apreferred embodiment the pullulanase includes an X47 domain as disclosedin U.S. 61/289,040 published as WO 2011/087836 (which are herebyincorporated by reference).

Specifically the pullulanase may be derived from a strain from the genusThermococcus, including Thermococcus litoralis and Thermococcushydrothermalis or a hybrid thereof.

The pullulanase may be Thermococcus hydrothermalis pullulanase truncatedat site X4 or a Thermococcus hydrothermalis/T. litoralis hybrid enzymewith truncation site X4 as disclosed in U.S. 61/289,040 published as WO2011/087836 or shown in SEQ ID NO: 12 herein.

In an embodiment the invention relates to a composition comprising:

i) an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂)of at least 10 derived from Bacillus stearothermophilus;

ii) a protease having a thermostability value of more than 20%determined as Relative Activity at 80° C./70° C. derived derived fromPyrococcus furiosus or Thermoascus aurantiacus; and optionally

iii) a glucoamylase derived from Penicillium oxalicum.

The Bacillus stearothermophilus alpha-amylase, Pyrococcus furiosus orThermoascus aurantiacus protease and/or Penicillium oxalicumglucoamylase may be any of the embodiment mentioned above.

In an embodiment the composition comprises a second alpha-amylase havinga T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of less than 10 derivedfrom Bacillus stearothermophilus.

In an embodiment the ratio of enzyme protein (weight basis) between thecomponents in a composition of the invention may be:

Alpha-Amylase: Glucoamylase: Protease: 0.1-10: 0.5-50: 0.1-7, such as0.5-3: 1-30: 0.5-2, such as 1-2: 5-20: 0.5-2.

Use of a Composition of the Invention

In a final aspect the invention relates to the use of a composition ofthe invention in a liquefaction process. In an embodiment liquefactionis a step in a process of the invention.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure, including definitions will becontrolling.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

Materials & Methods Materials:

Alpha-Amylase A: Bacillus stearothermophilus alpha-amylase with themutations I181*+G182*+N193F truncated to 491 amino acids (SEQ ID NO: 1)Alpha-Amylase 1093: Bacillus stearothermophilus alpha-amylase with themutations I181*+G182*+N193F+E129V+K177L+R179E truncated to 491 aminoacids (SEQ ID NO: 1)Alpha-Amylase 1407: Bacillus stearothermophilus alpha-amylase with themutationsI181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254Struncated to 491 amino acids (SEQ ID NO: 1)Alpha-Amylase 1236: Bacillus stearothermophilus alpha-amylase with themutations I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254Struncated to 491 amino acids (SEQ ID NO: 1)Protease 136: Metallo protease derived from Thermoascus aurantiacusCGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein andamino acids 1-177 in SEQ ID NO: 2 in WO 2003/048353 with the followingmutations: D79L+Y82F+S87P+A112P+A126V+D142LProtease 196: Metallo protease derived from Thermoascus aurantiacusCGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein andamino acids 1-177 in SEQ ID NO: 2 in WO 2003/048353 with the followingmutations: A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.Protease 077: Metallo protease derived from Thermoascus aurantiacusCGMCC No. 0670 disclosed as amino acids 1-177 in SEQ ID NO: 3 herein andamino acids 1-177 in SEQ ID NO: 2 in WO 2003/048353 with the followingmutations: A27K+D79L+S87P+A112P+D142L.Protease Pfu: Protease derived from Pyrococcus furiosus purchased fromTakara Bio Inc. (Japan) as Pfu Protease S (activity 10.5 mg/mL) and alsoshown in SEQ ID NO: 13 herein.Glucoamylase PO: Mature part of the Penicillium oxalicum glucoamylasedisclosed as SEQ ID NO: 2 in PCT/CN10/071753 published as WO 2011/127802and shown in SEQ ID NO: 9 and 14 herein.Glucoamylase PE001: Variant of the Penicillium oxalicum glucoamylasehaving a K79V substitution using the mature sequence shown in SEQ ID NO:14 for numbering.Glucoamylase BL: Blend of Tamaromyces emersonii glucoamylase disclosedin WO 99/28448 as SEQ ID NO: 7 and Trametes cingulata glucoamylasedisclosed in WO 06/069289 in a ratio of about 9:1.Glucoamylase BL2: Blend comprising Talaromyces emersonii glucoamylasedisclosed in WO 99/28448, Trametes cingulata glucoamylase disclosed inWO 06/69289, and Rhizomucor pusillus alpha-amylase with Aspergillusnigerglucoamylase linker and SBD disclosed as V039 in Table 5 in WO2006/069290 as side activities (ratio about 65:15:1) Substrate inExample 9: Ground corn from Corn LP, Iowa, USA (84.19% DS) and backset(6.27% DS).Pullulanase TH: Pullulanase from Thermococcus hydrothermalis shown inSEQ ID NO: 11 herein.Yeast: RED STAR ETHANOL RED™ available from Red Star/Lesaffre, USA.

Methods

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention the degree of identity between twoamino acid sequences, as well as the degree of identity between twonucleotide sequences, may be determined by the program “align” which isa Needleman-Wunsch alignment (i.e. a global alignment). The program isused for alignment of polypeptide, as well as nucleotide sequences. Thedefault scoring matrix BLOSUM50 is used for polypeptide alignments, andthe default identity matrix is used for nucleotide alignments. Thepenalty for the first residue of a gap is −12 for polypeptides and −16for nucleotides. The penalties for further residues of a gap are −2 forpolypeptides, and −4 for nucleotides.

“Align” is part of the FASTA package version v20u6 (see Pearson andLipman, 1988, “Improved Tools for Biological Sequence Analysis”, PNAS85:2444-2448, and Pearson, 1990, “Rapid and Sensitive SequenceComparison with FASTP and FASTA,” Methods in Enzymology 183:63-98).FASTA protein alignments use the Smith-Waterman algorithm with nolimitation on gap size (see “Smith-Waterman algorithm”, Smith andWaterman, 1981, J. Mol. Biol. 147:195-197).

Protease Assays AZCL-Casein Assay

A solution of 0.2% of the blue substrate AZCL-casein is suspended inBorax/NaH₂PO₄ buffer pH9 while stirring. The solution is distributedwhile stirring to microtiter plate (100 microL to each well), 30 microLenzyme sample is added and the plates are incubated in an EppendorfThermomixer for 30 minutes at 45° C. and 600 rpm. Denatured enzymesample (100° C. boiling for 20 min) is used as a blank. After incubationthe reaction is stopped by transferring the microtiter plate onto iceand the coloured solution is separated from the solid by centrifugationat 3000 rpm for 5 minutes at 4° C. 60 microL of supernatant istransferred to a microtiter plate and the absorbance at 595 nm ismeasured using a BioRad Microplate Reader.

pNA-Assay

50 microL protease-containing sample is added to a microtiter plate andthe assay is started by adding 100 microL 1 mM pNA substrate (5 mgdissolved in 100 microL DMSO and further diluted to 10 mL withBorax/NaH₂PO₄ buffer pH 9.0). The increase in OD₄₀₅ at room temperatureis monitored as a measure of the protease activity.

Glucoamylase Activity (AGU)

Glucoamylase activity may be measured in Glucoamylase Units (AGU).

The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme,which hydrolyzes 1 micromole maltose per minute under the standardconditions 37° C., pH 4.3, substrate: maltose 23.2 mM, buffer: acetate0.1 M, reaction time 5 minutes.

An autoanalyzer system may be used. Mutarotase is added to the glucosedehydrogenase reagent so that any alpha-D-glucose present is turned intobeta-D-glucose. Glucose dehydrogenase reacts specifically withbeta-D-glucose in the reaction mentioned above, forming NADH which isdetermined using a photometer at 340 nm as a measure of the originalglucose concentration.

AMG incubation: Substrate: maltose 23.2 mM Buffer: acetate 0.1M pH: 4.30± 0.05 Incubation temperature: 37° C. ± 1 Reaction time: 5 minutesEnzyme working range: 0.5-4.0 AGU/mL

Color reaction: GlucDH: 430 U/L Mutarotase:  9 U/L NAD: 0.21 mM Buffer:phosphate 0.12M; 0.15M NaCl pH: 7.60 ± 0.05 Incubation temperature: 37°C. ± 1 Reaction time: 5 minutes Wavelength: 340 nm

A folder (EB-SM-0131.02/01) describing this analytical method in moredetail is available on request from Novozymes A/S, Denmark, which folderis hereby included by reference.

Alpha-Amylase Activity (KNU)

The alpha-amylase activity may be determined using potato starch assubstrate. This method is based on the break-down of modified potatostarch by the enzyme, and the reaction is followed by mixing samples ofthe starch/enzyme solution with an iodine solution. Initially, ablackish-blue color is formed, but during the break-down of the starchthe blue color gets weaker and gradually turns into a reddish-brown,which is compared to a colored glass standard.

One Kilo Novo alpha amylase Unit (KNU) is defined as the amount ofenzyme which, under standard conditions (i.e., at 37° C.+/−0.05; 0.0003M Ca²⁺; and pH 5.6) dextrinizes 5260 mg starch dry substance MerckAmylum solubile.

A folder EB-SM-0009.02/01 describing this analytical method in moredetail is available upon request to Novozymes A/S, Denmark, which folderis hereby included by reference.

Determination of Pullulanase Activity (NPUN)

Endo-pullulanase activity in NPUN is measured relative to a Novozymespullulanase standard. One pullulanase unit (NPUN) is defined as theamount of enzyme that releases 1 micro mol glucose per minute under thestandard conditions (0.7% red pullulan (Megazyme), pH 5, 40° C., 20minutes). The activity is measured in NPUN/ml using red pullulan.

1 mL diluted sample or standard is incubated at 40° C. for 2 minutes.0.5 mL 2% red pullulan, 0.5 M KCl, 50 mM citric acid, pH 5 are added andmixed. The tubes are incubated at 40° C. for 20 minutes and stopped byadding 2.5 ml 80% ethanol. The tubes are left standing at roomtemperature for 10-60 minutes followed by centrifugation 10 minutes at4000 rpm. OD of the supernatants is then measured at 510 nm and theactivity calculated using a standard curve.

The present invention is described in further detail in the followingexamples which are offered to illustrate the present invention, but notin any way intended to limit the scope of the invention as claimed. Allreferences cited herein are specifically incorporated by reference forthat which is described therein.

EXAMPLES Example 1 Stability of Alpha-Amylase Variants

The stability of a reference alpha-amylase (Bacillus stearothermophilusalpha-amylase with the mutations I181*+G182*+N193F truncated to 491amino acids (SEQ ID NO: 1)) and alpha-amylase variants thereof wasdetermined by incubating the reference alpha-amylase and variants at pH4.5 and 5.5 and temperatures of 75° C. and 85° C. with 0.12 mM CaCl₂followed by residual activity determination using the EnzChek® substrate(EnzChek® Ultra Amylase assay kit, E33651, Molecular Probes).

Purified enzyme samples were diluted to working concentrations of 0.5and 1 or 5 and 10 ppm (micrograms/ml) in enzyme dilution buffer (10 mMacetate, 0.01% Triton X100, 0.12 mM CaCl₂, pH 5.0). Twenty microlitersenzyme sample was transferred to 48-well PCR MTP and 180 microlitersstability buffer (150 mM acetate, 150 mM MES, 0.01% Triton X100, 0.12 mMCaCl₂, pH 4.5 or 5.5) was added to each well and mixed. The assay wasperformed using two concentrations of enzyme in duplicates. Beforeincubation at 75° C. or 85° C., 20 microliters was withdrawn and storedon ice as control samples. Incubation was performed in a PCR machine at75° C. and 85° C. After incubation samples were diluted to 15 ng/mL inresidual activity buffer (100 mM Acetate, 0.01% Triton X100, 0.12 mMCaCl₂, pH 5.5) and 25 microliters diluted enzyme was transferred toblack 384-MTP. Residual activity was determined using the EnzCheksubstrate by adding 25 microliters substrate solution (100micrograms/ml) to each well. Fluorescence was determined every minutefor 15 minutes using excitation filter at 485-P nm and emission filterat 555 nm (fluorescence reader is Polarstar, BMG). The residual activitywas normalized to control samples for each setup.

Assuming logarithmic decay half life time (T½ (min)) was calculatedusing the equation: T½ (min)=T(min)*LN(0.5)/LN(% RA/100), where T isassay incubation time in minutes, and % RA is % residual activitydetermined in assay.

Using this assay setup the half life time was determined for thereference alpha-amylase and variant thereof as shown in Table 1.

TABLE 1 T½ (min) T½ (min) T½ (min) (pH 4.5, 75° C., (pH 4.5, 85° C., (pH5.5, 85° C., Mutations 0.12 mM CaCl₂) 0.12 mM CaCl₂) 0.12 mM CaCl₂)Reference Alpha-Amylase A 21 4 111 Reference Alpha-Amylase A with 32 6301 the substitution V59A Reference Alpha-Amylase A with 28 5 230 thesubstitution V59E Reference Alpha-Amylase A with 28 5 210 thesubstitution V59I Reference Alpha-Amylase A with 30 6 250 thesubstitution V59Q Reference Alpha-Amylase A with 149 22 ND thesubstitutions V59A + Q89R + G112D + E129V + K177L + R179E + K220P +N224L + Q254S Reference Alpha-Amylase A with >180 28 ND thesubstitutions V59A + Q89R + E129V + K177L + R179E + H208Y + K220P +N224L + Q254S Reference Alpha-Amylase A with 112 16 ND the substitutionsV59A + Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + D269E +D281N Reference Alpha-Amylase A with 168 21 ND the substitutions V59A +Q89R + E129V + K177L + R179E + K220P + N224L + Q254S + I270L ReferenceAlpha-Amylase A with >180 24 ND the substitutions V59A + Q89R + E129V +K177L + R179E + K220P + N224L + Q254S + H274K Reference Alpha-Amylase Awith 91 15 ND the substitutions V59A + Q89R + E129V + K177L + R179E +K220P + N224L + Q254S + Y276F Reference Alpha-Amylase A with 141 41 NDthe substitutions V59A + E129V + R157Y + K177L + R179E + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 62 ND thesubstitutions V59A + E129V + K177L + R179E + H208Y + K220P + N224L +S242Q + Q254S Reference Alpha-Amylase A with >180 49 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S Reference Alpha-Amylase A with >180 53 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + S242Q + Q254S + H274K ReferenceAlpha-Amylase A with >180 57 ND the substitutions V59A + E129V + K177L +R179E + K220P + N224L + S242Q + Q254S + Y276F Reference Alpha-Amylase Awith >180 37 ND the substitutions V59A + E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + D281N Reference Alpha-Amylase A with >180 51 NDthe substitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + M284T Reference Alpha-Amylase A with >180 45 ND thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + G416V Reference Alpha-Amylase A with 143 21 >480 thesubstitutions V59A + E129V + K177L + R179E + K220P + N224L + Q254SReference Alpha-Amylase A with >180 22 ND the substitutions V59A +E129V + K177L + R179E + K220P + N224L + Q254S + M284T ReferenceAlpha-Amylase A with >180 38 ND the substitutions A91L + M96I + E129V +K177L + R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 57 11 402 the substitutions E129V + K177L + R179E ReferenceAlpha-Amylase A with 174 44 >480 the substitutions E129V + K177L +R179E + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + Y276F + L427M Reference Alpha-Amylase Awith >180 49 >480 the substitutions E129V + K177L + R179E + K220P +N224L + S242Q + Q254S + M284T Reference Alpha-Amylase A with 177 36 >480the substitutions E129V + K177L + R179E + K220P + N224L + S242Q +Q254S + N376* + I377* Reference Alpha-Amylase A with 94 13 >480 thesubstitutions E129V + K177L + R179E + K220P + N224L + Q254S ReferenceAlpha-Amylase A with 129 24 >480 the substitutions E129V + K177L +R179E + K220P + N224L + Q254S + M284T Reference Alpha-Amylase A with 14830 >480 the substitutions E129V + K177L + R179E + S242Q ReferenceAlpha-Amylase A with 78 9 >480 the substitutions E129V + K177L + R179VReference Alpha-Amylase A with 178 31 >480 the substitutions E129V +K177L + R179V + K220P + N224L + S242Q + Q254S Reference Alpha-Amylase Awith 66 17 >480 the substitutions K220P + N224L + S242Q + Q254SReference Alpha-Amylase A with 30 6 159 the substitutions K220P +N224L + Q254S Reference Alpha-Amylase A with 35 7 278 the substitutionM284T Reference Alpha-Amylase A with 59 13 ND the substitutions M284V NDnot determined

The results demonstrate that the alpha-amylase variants have asignificantly greater half-life and stability than the referencealpha-amylase.

Example 2 Preparation of Protease Variants and Test of Thermostability

Chemicals used were commercial products of at least reagent grade.

Strains and Plasmids:

E. coli DH12S (available from Gibco BRL) was used for yeast plasmidrescue. pJTP000 is a S. cerevisiae and E. coli shuttle vector under thecontrol of TPI promoter, constructed from pJC039 described in WO01/92502, in which the Thermoascus aurantiacus M35 protease gene (WO03/048353) has been inserted.

Saccharomyces cerevisiae YNG318 competent cells: Mata Dpep4[cir+]ura3-52, leu2-D2, his 4-539 was used for protease variants expression.It is described in J. Biol. Chem. 272(15): 9720-9727 (1997).

Media and Substrates

10× Basal solution: Yeast nitrogen base w/o amino acids (DIFCO) 66.8g/L, succinate 100 g/l, NaOH 60 g/l.SC-glucose: 20% glucose (i.e., a final concentration of 2%=2 g/100 mL))100 mL/L, 5% threonine 4 mL/L, 1% tryptophan 10 ml/l, 20% casamino acids25 ml/l, 10× basal solution 100 ml/l. The solution is sterilized using afilter of a pore size of 0.20 micrometer. Agar (2%) and H₂O (approx. 761mL) is autoclaved together, and the separately sterilized SC-glucosesolution is added to the agar solution.YPD: Bacto peptone 20 g/l, yeast extract 10 g/L, 20% glucose 100 mL/L.

YPD+Zn: YPD+0.25 mM ZnSO₄.

PEG/LiAc solution: 40% PEG4000 50 ml, 5 M Lithium Acetate 1 mL.96 well Zein micro titre plate:

Each well contains 200 microL of 0.05-0.1% of zein (Sigma), 0.25 mMZnSO₄ and 1% of agar in 20 mM sodium acetate buffer, pH 4.5.

DNA Manipulations

Unless otherwise stated, DNA manipulations and transformations wereperformed using standard methods of molecular biology as described inSambrook et al. (1989) Molecular cloning: A laboratory manual, ColdSpring Harbor lab. Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.) “Current protocols in Molecular Biology”, John Wiley and Sons,1995; Harwood, C. R. and Cutting, S. M. (Eds.).

Yeast Transformation

Yeast transformation was performed using the lithium acetate method. 0.5microL of vector (digested by restriction endnucleases) and 1 microL ofPCR fragments is mixed. The DNA mixture, 100 microL of YNG318 competentcells, and 10 microL of YEAST MAKER carrier DNA (Clontech) is added to a12 mL polypropylene tube (Falcon 2059). Add 0.6 mL PEG/LiAc solution andmix gently. Incubate for 30 min at 30° C., and 200 rpm followed by 30min at 42° C. (heat shock). Transfer to an eppendorf tube and centrifugefor 5 sec. Remove the supernatant and resolve in 3 mL of YPDv. Incubatethe cell suspension for 45 min at 200 rpm at 30° C. Pour the suspensionto SC-glucose plates and incubate 30° C. for 3 days to grow colonies.Yeast total DNA are extracted by Zymoprep Yeast Plasmid Miniprep Kit(ZYMO research).

DNA Sequencing

E. coli transformation for DNA sequencing was carried out byelectroporation (BIO-RAD Gene Pulser). DNA Plasmids were prepared byalkaline method (Molecular Cloning, Cold Spring Harbor) or with theQiagen® Plasmid Kit. DNA fragments were recovered from agarose gel bythe Qiagen gel extraction Kit. PCR was performed using a PTC-200 DNAEngine. The ABI PRISM™ 310 Genetic Analyzer was used for determinationof all DNA sequences.

Construction of Protease Expression Vector

The Themoascus M35 protease gene was amplified with the primer pair ProtF (SEQ ID NO: 4) and Prot R (SEQ ID NO: 5). The resulting PCR fragmentswere introduced into S. cerevisiae YNG318 together with the pJC039vector (described in WO 2001/92502) digested with restriction enzymes toremove the Humicola insolens cutinase gene.

The Plasmid in yeast clones on SC-glucose plates was recovered toconfirm the internal sequence and termed as pJTP001.

Construction of Yeast Library and Site-Directed Variants

Library in yeast and site-directed variants were constructed by SOE PCRmethod (Splicing by Overlap Extension, see “PCR: A practical approach”,p. 207-209, Oxford University press, eds. McPherson, Quirke, Taylor),followed by yeast in vivo recombination.

General Primers for Amplification and Sequencing

The primers AM34 (SEQ ID NO: 6) and AM35 (SEQ ID NO:7) were used to makeDNA fragments containing any mutated fragments by the SOE methodtogether with degenerated primers (AM34+Reverse primer and AM35+forwardprimer) or just to amplify a whole protease gene (AM34+AM35).

PCR reaction system: Conditions: 48.5 microL H₂O 1 94° C. 2 min 2 beadspuRe Taq Ready-To-Go PCR 2 94° C. 30 sec (Amersham Biosciences) 0.5microL × 2 100 pmole/microL of primers 3 55° C. 30 sec 0.5 microLtemplate DNA 4 72° C. 90 sec 2-4 25 cycles 5 72° C. 10 min

DNA fragments were recovered from agarose gel by the Qiagen gelextraction Kit. The resulting purified fragments were mixed with thevector digest. The mixed solution was introduced into Saccharomycescerevisiae to construct libraries or site-directed variants by in vivorecombination.

Relative Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate containing YPD+Zn medium and cultivated at 28° C. for 3days. The culture supernatants were applied to a 96-well zein microtiter plate and incubated at at least 2 temperatures (ex., 70° C. and80° C.) for more than 4 hours or overnight. The turbidity of zein in theplate was measured as A630 and the relative activity (higher/lowertemperatures) was determined as an indicator of thermoactivityimprovement. The clones with higher relative activity than the parentalvariant were selected and the sequence was determined.

Remaining Activity Assay

Yeast clones on SC-glucose were inoculated to a well of a 96-well microtitre plate and cultivated at 28° C. for 3 days. Protease activity wasmeasured at 65° C. using azo-casein (Megazyme) after incubating theculture supernatant in 20 mM sodium acetate buffer, pH 4.5, for 10 minat a certain temperature (80° C. or 84° C. with 4° C. as a reference) todetermine the remaining activity. The clones with higher remainingactivity than the parental variant were selected and the sequence wasdetermined.

Azo-Casein Assay

20 microL of samples were mixed with 150 microL of substrate solution (4mL of 12.5% azo-casein in ethanol in 96 mL of 20 mM sodium acetate, pH4.5, containing 0.01% triton-100 and 0.25 mM ZnSO₄) and incubated for 4hours or longer.

After adding 20 microL/well of 100% trichloroacetic acid (TCA) solution,the plate was centrifuge and 100 microL of supernatants were pipette outto measure A440.

Expression of Protease Variants in Aspergillus oryzae

The constructs comprising the protease variant genes were used toconstruct expression vectors for Aspergillus. The Aspergillus expressionvectors consist of an expression cassette based on the Aspergillus nigerneutral amylase II promoter fused to the Aspergillus nidulans triosephosphate isomerase non translated leader sequence (Pna2/tpi) and theAspergillus niger amyloglycosidase terminator (Tamg). Also present onthe plasmid was the Aspergillus selective marker amdS from Aspergillusnidulans enabling growth on acetamide as sole nitrogen source. Theexpression plasmids for protease variants were transformed intoAspergillus as described in Lassen et al., 2001, Appl. Environ.Microbiol. 67: 4701-4707. For each of the constructs 10-20 strains wereisolated, purified and cultivated in shake flasks.

Purification of Expressed Variants

-   1. Adjust pH of the 0.22 μm filtered fermentation sample to 4.0.-   2. Put the sample on an ice bath with magnetic stirring. Add    (NH₄)₂SO₄ in small aliquots (corresponding to approx. 2.0-2.2 M    (NH₄)₂SO₄ not taking the volume increase into account when adding    the compound).-   3. After the final addition of (NH₄)₂SO₄, incubate the sample on the    ice bath with gentle magnetic stirring for min. 45 min.-   4. Centrifugation: Hitachi himac CR20G High-Speed Refrigerated    Centrifuge equipped with R20A2 rotor head, 5° C., 20,000 rpm, 30    min.-   5. Dissolve the formed precipitate in 200 mL 50 mM Na-acetate pH    4.0.-   6. Filter the sample by vacuum suction using a 0.22 micro m PES PLUS    membrane (IWAKI).-   7. Desalt/buffer-exchange the sample to 50 mM Na-acetate pH 4.0    using ultrafiltration (Vivacell 250 from Vivascience equipped with 5    kDa MWCO PES membrane) overnight in a cold room. Dilute the    retentate sample to 200 ml using 50 mM Na-acetate pH 4.0. The    conductivity of sample is preferably less than 5 mS/cm.-   8. Load the sample onto a cation-exchange column equilibrated with    50 mM Na-acetate pH 4.0. Wash unbound sample out of the column using    3 column volumes of binding buffer (50 mM Na-acetate pH 4.0), and    elute the sample using a linear gradient, 0-100% elution buffer (50    mM Na-acetate+1 M NaCl pH 4.0) in 10 column volumes.-   9. The collected fractions are assayed by an endo-protease assay    (cf. below) followed by standard SDS-PAGE (reducing conditions) on    selected fractions. Fractions are pooled based on the endo-protease    assay and SDS-PAGE.

Endo-Protease Assay

-   1. Protazyme OL tablet/5 ml 250 mM Na-acetate pH 5.0 is dissolved by    magnetic stirring (substrate: endo-protease Protazyme AK tablet from    Megazyme—cat. #PRAK 11/08).-   2. With stirring, 250 microL of substrate solution is transferred to    a 1.5 mL Eppendorf tube.-   3. 25 microL of sample is added to each tube (blank is sample    buffer).-   4. The tubes are incubated on a Thermomixer with shaking (1000 rpm)    at 50° C. for 15 minutes.-   5. 250 microL of 1 M NaOH is added to each tube, followed by    vortexing.-   6. Centrifugation for 3 min. at 16,100×G and 25° C.-   7. 200 microL of the supernatant is transferred to a MTP, and the    absorbance at 590 nm is recorded.

TABLE 2 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Remaining Activity VariantSubstitution(s) and/or deletion(s) 80° C. 84° C. JTP082ΔS5/D79L/S87P/A112P/D142L 53% JTP091 D79L/S87P/A112P/T124V/D142L 43%JTP092 ΔS5/N26R/D79L/S87P/A112P/D142L 60% JTP095N26R/T46R/D79L/S87P/A112P/D142L 62% JTP096 T46R/D79L/S87P/T116V/D142L67% JTP099 D79L/P81R/S87P/A112P/D142L 80% JTP101A27K/D79L/S87P/A112P/T124V/D142L 81% JTP116D79L/Y82F/S87P/A112P/T124V/D142L 59% JTP117D79L/Y82F/S87P/A112P/T124V/D142L 94% JTP127D79L/S87P/A112P/T124V/A126V/D142L 53%

TABLE 3 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Relative Activity Variant Substitutions80° C./70° C. 85° C./70° C. JTP050 D79L S87P A112P D142L 23%  9% JTP134D79L Y82F S87P A112P D142L 40% JTP135 S38T D79L S87P A112P A126V D142L62% JTP136 D79L Y82F S87P A112P A126V D142L 59% JTP137 A27K D79L S87PA112P A126V D142L 54% JTP145 S49P D79L S87P A112P D142L 59% JTP146 S50PD79L S87P A112P D142L 63% JTP148 D79L S87P D104P A112P D142L 64% JTP161D79L Y82F S87G A112P D142L 30% 12% JTP180 S70V D79L Y82F S87G Y97W A112PD142L 52% JTP181 D79L Y82F S87G Y97W D104P A112P D142L 45% JTP187 S70VD79L Y82F S87G A112P D142L 45% JTP188 D79L Y82F S87G D104P A112P D142L43% JTP189 D79L Y82F S87G A112P A126V D142L 46% JTP193 Y82F S87G S70VD79L D104P A112P D142L 15% JTP194 Y82F S87G D79L D104P A112P A126V D142L22% JTP196 A27K D79L Y82F S87G D104P A112P A126V D142L 18%

TABLE 4 Relative Activity of protease variants. Numbering ofsubstitution(s) starts from N-terminal of the mature peptide in aminoacids 1 to 177 of SEQ ID NO: 3. Relative Activity Variant Substitutions80° C./70° C. JTP196 A27K D79L Y82F S87G D104P A112P A126V D142L 55%JTP210 A27K Y82F S87G D104P A112P A126V D142L 36% JTP211 A27K D79L Y82FD104P A112P A126V D142L 44% JTP213 A27K Y82F D104P A112P A126V D142L 37%

Example 3 Temperature Profile of Selected Protease Variants UsingPurified Enzymes

Selected protease variants showing good thermostability were purifiedand the purified enzymes were used in a zein-BCA assay as describedbelow. The remaining protease activity was determined at 60° C. afterincubation of the enzyme at elevated temperatures as indicated for 60min.

Zein-BCA Assay:

Zein-BCA assay was performed to detect soluble protein quantificationreleased from zein by variant proteases at various temperatures.

Protocol:

-   1) Mix 10 microL of 10 micro g/mL enzyme solutions and 100 microL of    0.025% zein solution in a micro titer plate (MTP).-   2) Incubate at various temperatures for 60 min.-   3) Add 10 microL of 100% trichloroacetic acid (TCA) solution.-   4) Centrifuge MTP at 3500 rpm for 5 min.-   5) Take out 15 microL to a new MTP containing 100 microL of BCA    assay solution (Pierce Cat #:23225, BCA Protein Assay Kit).-   6) Incubate for 30 min. at 60° C.-   7) Measure A562.

The results are shown in Table 5. All of the tested protease variantsshowed an improved thermostability as compared to the wild type (WT)protease.

TABLE 5 Zein-BCA assay Sample incubated 60 min at indicated temperatures(° C.) (micro g/mL Bovine serum albumin equivalent peptide released)WT/Variant 60° C. 70° C. 75° C. 80° C. 85° C. 90° C. 95° C. WT 94 103107 93 58 38 JTP050 86 101 107 107 104 63 36 (D79L + S87P + A112P +D142L) JTP077 82 94 104 105 99 56 31 (A27K + D79L + S87P + A112P +D142L) JTP188 71 83 86 93 100 75 53 (D79L + Y82F + S87G + D104P +A112P + D142L) JTP196 87 99 103 106 117 90 38 (A27K + D79L + Y82F +S87G + D104P + A112P + A126V + D142L)

Example 4

Characterization of Penicillium oxalicum Glucoamylase

The Penicillium oxalicum glucoamylase is disclosed WO 2011/127802 and inSEQ ID NO: 9 herein.

Substrate. Substrate: 1% soluble starch (Sigma S-9765) in deionizedwater

Reaction buffer: 0.1 M Acetate buffer at pH 5.3

Glucose concentration determination kit: Wako glucose assay kit(LabAssay glucose, WAKO, Cat #298-65701).

Reaction condition. 20 microL soluble starch and 50 microL acetatebuffer at pH5.3 were mixed. 30 microL enzyme solution (50 micro g enzymeprotein/ml) was added to a final volume of 100 microL followed byincubation at 37° C. for 15 min.

The glucose concentration was determined by Wako kits.

All the work carried out in parallel.

Temperature optimum. To assess the temperature optimum of thePenicillium oxalicum glucoamylase the “Reaction condition”-assaydescribed above was performed at 20, 30, 40, 50, 60, 70, 80, 85, 90 and95° C. The results are shown in Table 6.

TABLE 6 Temperature optimum Temperature (° C.) 20 30 40 50 60 70 80 8590 95 Relative 63.6 71.7 86.4 99.4 94.6 100.0 92.9 92.5 82.7 82.8activity (%)

From the results it can be seen that the optimal temperature forPenicillium oxalicum glucoamylase at the given conditions is between 50°C. and 70° C. and the glucoamylase maintains more than 80% activity at95° C.

Heat stability. To assess the heat stability of the Penicillium oxalicumglucoamylase the Reaction condition assay was modified in that the theenzyme solution and acetate buffer was preincubated for 15 min at 20,30, 40, 50, 60, 70, 75, 80, 85, 90 and 95° C. Following the incubation20 microL of starch was added to the solution and the assay wasperformed as described above.

The results are shown in Table 7.

TABLE 7 Heat stability Temperature (° C.) 20 30 40 50 60 70 80 85 90 95Relative 91.0 92.9 88.1 100.0 96.9 86.0 34.8 36.0 34.2 34.8 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseis stable up to 70° C. after preincubation for 15 min in that itmaintains more than 80% activity.

pH optimum. To assess the pH optimum of the Penicillium oxalicumglucoamylase the Reaction condition assay described above was performedat pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0.Instead of using the acetate buffer described in the Reaction conditionassay the following buffer was used 100 mM Succinic acid, HEPES, CHES,CAPSO, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH adjusted to 2.0,3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 or 11.0 with HCl orNaOH.

The results are shown in Table 8.

TABLE 8 pH optimum pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.0 11.0Relative 71.4 78.6 77.0 91.2 84.2 100.0 55.5 66.7 30.9 17.8 15.9 16.1activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylaseat the given conditions has the highest activity at pH 5.0. ThePenicillium oxalicum glucoamylase is active in a broad pH range in theit maintains more than 50% activity from pH 2 to 7.

pH stability. To assess the heat stability of the Penicillium oxalicumglucoamylase the Reaction condition assay was modified in that theenzyme solution (50 micro g/mL) was preincubated for 20 hours in bufferswith pH 2.0, 3.0, 3.5, 4.0, 4.5, 5.0, 6.0 7.0, 8.0, 9.0, 10.0 and 11.0using the buffers described under pH optimum. After preincubation, 20microL soluble starch to a final volume of 100 microL was added to thesolution and the assay was performed as described above.

The results are shown in Table 9.

TABLE 9 pH stability pH 2.0 3.0 3.5 4.0 4.5 5.0 6.0 7.0 8.0 9.0 10.011.0 Relative 17.4 98.0 98.0 103.2 100.0 93.4 71.2 90.7 58.7 17.4 17.017.2 activity (%)

From the results it can be seen that Penicillium oxalicum glucoamylase,is stable from pH 3 to pH 7 after preincubation for 20 hours and itdecreases its activity at pH 8.

Example 5 Improved Ethanol Production Process

Mash Preparation: Corn mashes were prepared through liquefaction in an85° C. water bath for 2 hours. The dry solids (DS) content was around30-33% and the backset ratio around 30%.Mash Preparation: Corn slurries were prepared for liquefaction byweighing out the specified amounts of ground corn, backset, and tapwater into Nalgene bottles. Slurries were pH-adjusted to either 5.80(Control) using 50% w/w NaOH or 4.50 (Study A, B) or 4.80 (Study C, D,E, F) using 40% v/v H₂SO₄. Control mashes using Alpha-Amylase A weremade at pH 5.8. Aliquots of enzyme stock solutions were added. Bottleswere tightly capped and placed into the water bath. Slurries were shakenvigorously once every 5 minutes for the first 30 minutes and then onceevery 30 minutes thereafter for a total of 2 hours. Mashes wereimmediately cooled in an ice bath. Urea and penicillin were then addedto each mash to reach concentrations of 500 and 3 ppm, respectively.Fermentation Setup: Mashes were adjusted to pH 5.0 using 40% H₂SO₄ or50% NaOH. Approximately 5 g of each mash was transferred intopre-weighed 15 mL plastic Falcon centrifuge tubes for fermentation.Typically, five replicate fermentations were prepared for eachtreatment. A small hole was drilled into the lid of each tube to allowfor CO₂ release during fermentation. Following mash transfer, all tubeswere reweighed to obtain their initial sample weights. Into each tubewas then added 100 microL of rehydrated RED STAR ETHANOL RED yeast(rehydrated by weighing 5.5 g of dry yeast into a 150 mL Erlenmeyerflask, adding 100 mL of tap water and stirring in a 32° C. water bathfor 30 minutes), an aliquot of diluted Glucoamylase BL (diluted indeionized water) needed to reach starting concentrations of 0.50 AGU/gDS. Deionized water was added to each tube such that the total volume ofliquid added to each tube relative to the sample weight was the same.All tubes were then reweighed and then placed into a water bath set at32° C. Fermentation was typically allowed to progress for 54 hours (ifnothing else is stated). Tubes were vigorously vortexed afterapproximately 7 hours and then vortexed and reweighed twice per day forthe remaining fermentation time. The grams of ethanol produced per gramof dry solids in each tube were calculated from the weight loss dataaccording to the following equation:

${g\mspace{14mu} {ethanol}\text{/}g\mspace{14mu} {DS}} = \frac{\begin{matrix}{g\mspace{14mu} {CO}_{2}\mspace{14mu} {weight}\mspace{14mu} {loss} \times \frac{1\mspace{14mu} {mol}\mspace{14mu} {CO}_{2}}{44.0098\mspace{14mu} g\mspace{14mu} {CO}_{2}} \times} \\{\frac{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {CO}_{2}} \times \frac{46.094\mspace{14mu} g\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}}\end{matrix}}{g\mspace{14mu} {corn}\mspace{14mu} {in}\mspace{14mu} {tube} \times \% {DS}\mspace{14mu} {of}\mspace{14mu} {corn}}$

Typically, 4 replicate tubes for each treatment were pulled after 54hours of fermentation for HPLC analysis. Pulled samples were treatedwith 50 microL of 40% H₂SO₄ to stop fermentation and vortexedthoroughly. The samples were then centrifuged at 1460×g for 10 minutesand then filtered into HPLC vials through 0.45 micro m syringe filters.HPLC analysis was finally conducted on the samples to quantify theamounts of ethanol.

Results

An overview of the results is provided in Table 10.

TABLE 10 The doses of enzymes are listed in parentheses for each and areexpressed as micro g EP/g DS. HPLC EtOH vs Reference Alpha- Enzymes InGlucoamylase Amylase A Study pH liquefaction step i). In SSF (Control) A4.5 Alpha-Amylase 1093 (1.4) Glucoamylase BL 3.0% Protease 077 (2)Pullulanase TH (2) B 4.5 Alpha-amylase 1093 (2.75) Glucoamylase BL 1.6%Protease 077 (5) Pullulanase TH (2) C 4.8 Alpha-amylase 1236 (2)Glucoamylase BL 4.7% Protease 136 (2) Glucoamylase PO (15) D 4.8Alpha-Amylase 1093 (2) Glucoamylase BL 4.2% (48 hrs) Protease 180 (2, 1)Glucoamylase PO (10) E 4.8 Alpha-Amylase 1236 (2) Glucoamylase BL 7.1%Protease 188 (2) Glucoamylase PO (15) F 4.8 Alpha-Amylase 1407 (1)Glucoamylase BL 4.8% (at 72 hrs) Protease 196 (2) Glucoamylase PO (2)*measured at 54 hours unless otherwise noted.

Example 6

Whole Corn Liquefaction and SSF Process Using the P. oxalicum AMGVariant (PE001)

The Penicillium oxalicum glucoamylase (Glucoamylase PO) variant,Glucoamylase PE001, showing reduced sensitivity to protease degradation,was tested in both whole corn liquefaction and starch saccharification(shown in next section). For the whole corn liquefactions, theGlucoamylase PE001 enzyme was added in different doses with a low pHamylase variant, Alpha-Amylase 1407. In some liquefactions, theGlucoamylase PE001 variant was tested with both the low pH amylaseAlpha-Amylase 1407 and the thermostable protease Protease 196. In allexperiments, the liquefactions were done using the automated systemcalled the “Lab-O-Mat”. This instrument controls the temperature andprovides constant mixing. The other experimental conditions were: pH was4.8 (for the liquefacts containing the Alpha-Amylase 1407 low pHamylase) or 5.8 (for the Alpha-Amylase A control), 32% dry solids, 85°C., 2 hours total time. The enzyme dosing schemes are shown in Table 11.The liquefied mashes were saccharified and fermented using GlucoamylaseBL2 (at a dose of 0.5 AGU/gram dry solids for 54 hours at 32° C.).

TABLE 11 Enzyme dosing scheme for the three whole corn liquefactionexperiments done using Glucoamylase PO protease nicking stable variant,i.e., Glucoamylase PE001. Alpha-Amylase (Dose) Protease (Dose)Glucoamylase (Dose) Alpha-Amylase A None None (0.02% w/w corn)Alpha-Amylase 1407 None None (1.4 μg EP/g DS) Alpha-Amylase 1407 NoneGlucoamylase PO (1.4 μg EP/g DS) (P3HK) (10 μg EP/g DS) Alpha-Alpha 1407None Glucoamylase PE001 (1.4 μg EP/g DS) (10 μg EP/g DS) Alpha-Amylase1407 Protease 196 Glucoamylase PO (1.4 μg EP/g DS) (1 μg EP/g DS) (P3HK)(10 μg EP/g DS) Alpha-Amylase 1407 Protease 196 Glucoamylase PE001 (1.4μg EP/g DS) (1 μg EP/g DS) (10 μg EP/g DS)

The HPLC quantified ethanol titers (in grams per liter) are shown inTable 12.

TABLE 12 Average ethanol titers and associated standard deviations, ingrams per liter. The Protease196 is a temperature stable proteasedescribed in WO 2011/072191 and Alpha-Amylase 1407 is a low pH amylasedescribed in WO 2011/082425. Ethanol (Average ± Standard Treatmentdeviation; grams/liter) Alpha-Amylase A control 126.4 ± 0.3Alpha-Amylase 1407 126.7 ± 0.3 (low pH alpha-amylase variant) controlGlucoamylase PO (wild-type) P3HK 127.2 ± 0.4 (10 μg EP/g DS)Glucoamylase PE001 variant (10 μg EP/g DS) 127.1 ± 0.5 Glucoamylase PO(wild-type) P3HK 127.6 ± 0.4 (10 μg EP/g DS) + Protease 196 (1 μg EP/gDS) Glucoamylase PE001 variant (10 μg EP/g DS) + 127.7 ± 0.2 Protease196 (1 μg EP/g DS)

Example 7 Thermostability of Protease Pfu

The thermostability of the Pyrococcus furiosus protease (Pfu S)purchased from Takara Bio, (Japan) was tested using the same methods asin Example 2. It was found that the thermostability (Relative Activity)was 110% at (80° C./70° C.) and 103% (90° C./70° C.) at pH 4.5.

Example 8 Ethanol Production Using Alpha-Amylase 1407 and Pfu Proteasefor Liquefaction

The purpose of this experiment was to evaluate application performanceof Protease Pfu derived from Pyrococcus furiosus at pH 4.8 duringliquefaction at 85° C. for 2 hours. Liquefaction (Labomat)

Each liquefaction received ground corn (84.19% DS), backset (6.27% DS),and tap water targeting a total weight of 100 g at 32.50% Dry Solids(DS). Backset was blended at 30% w/w of total slurry weight. Initialslurry pH was approximately 5.2 and was adjusted to pH 4.8 with 40% v/vsulfuric acid prior to liquefaction. All enzymes were added according tothe experimental design listed in Table 13 below. Liquefaction tookplace in a Labomat using the following conditions: 5° C./min. Ramp, 17minute Ramp, 103 minute hold time, 40 rpm for the entire run, 200 mLstainless steel canisters. After liquefaction, all canisters were cooledin an ice bath and prepared for fermentation based on the protocollisted below under SSF.

Simultaneous Saccharification and Fermentation (SSF)

Each mash was adjusted to pH 5.0 with 50% w/w Sodium Hydroxide or 40%v/v sulfuric acid. Penicillin was applied to each mash to a totalconcentration of 3 ppm. The tubes were prepared with mash by aliquotingapproximately 4.5 g of mash per 15 mL pre-drilled test tubes to allowCO₂ release. The test tubes sat, overnight, at 4° C. until the nextmorning.

All test tubes of mash were removed from cold storage and warmed up to32° C. in the walk-in incubation chamber. Once warmed, Glucoamylase BL2,was dosed to each tube of mash at 0.50 AGU/g DS, water was added so thatall tubes received 120 μL of liquid and each mash sample received 100 μLof rehydrated yeast. Rehydrated yeast was prepared by mixing 5.5 g ofFermentis RED STAR into 100 mL of 32° C. tap water for at least 15minutes.

In monitoring CO₂ weight-loss over time, each unit of CO₂ generated andlost is converted to gram ethanol produced per gram of dry solids (gEtOH/gDS) by the following:

${g\mspace{14mu} {ethanol}\text{/}g\mspace{14mu} {DS}} = \frac{\begin{matrix}{g\mspace{14mu} {CO2}\mspace{14mu} {weight}\mspace{14mu} {loss} \times \frac{1\mspace{14mu} {mol}\mspace{14mu} {CO2}}{44.0098\mspace{14mu} g\mspace{14mu} {CO2}}} \\{\frac{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {CO2}}\frac{46.094\mspace{14mu} g\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}}\end{matrix}}{g\mspace{14mu} {mash}\mspace{14mu} {in}\mspace{14mu} {tube}\mspace{14mu} \% {DS}\mspace{14mu} {of}\mspace{14mu} {mash}}$

HPLC Analysis

Fermentation sampling took place after 54 hours of fermentation bytaking 3 tubes per treatment. Each sample was deactivated with 50 μL of40% v/v H₂SO₄, vortexing, centrifuging at 1460×g for 10 minutes, andfiltering through a 0.45 μm Whatman PP filter. 54 hour samples wereanalyzed under HPLC without further dilution. Samples were stored at 4°C. prior to and during HPLC analysis.

HPLC Agilent's 1100/1200 series with Chem station software systemDegasser, Quaternary Pump, Auto-Sampler, Column Compartment/w HeaterRefractive Index Detector (RI) Column Bio-Rad HPX-87H Ion ExclusionColumn 300 mm × 7.8 mm part# 125-0140 Bio-Rad guard cartridge cation Hpart# 125-0129, Holder part# 125-0131 Method 0.005M H₂SO₄ mobile phaseFlow rate: 0.6 ml/min Column temperature: 65° C. RI detectortemperature: 55° C.

The method quantified analyte(s) using calibration standard for ethanol(% w/v). A four point calibration including the origin is used forquantification.

Where applicable, data were analyzed using JMP software (Cary, N.C.)with Oneway ANOVA of pairs using Tukey-Kramer HSD or Dunnett's. Errorbars denoting the 95% confidence level were established by multiplyingthe standard error of Oneway Anova analysis by 1.96.

TABLE 13 Experimental Plan. Liquefaction at 85° C. (pH 4.8) Alpha- DoseDose Dose amylase μg/g DS Protease μg/g DS Glucoamylase μg/g DS 1407 1.4— — — — 1407 1.4 Pfu 2 — — 1407 1.4 Pfu 2 PE001 10

Table 14 and FIG. 1 below show the results:

EtOH EtOH JMP Std 95% Treatment pH (% w/v) (% Δ) Error CI Control 4.8 9.2 100% 0.022 0.042 Pfu 4.8 11.0 120% 0.022 0.042 Pfu + PE001 4.8 11.0120% 0.022 0.042

Example 9 Ethanol Production Using Alpha-Amylase 1407 and Pfu Proteasefor Liquefaction

The purpose of this experiment was to evaluate application performanceof Protease Pfu derived from Pyrococcus furiosus at pH 4.8 duringliquefaction at 85° C. for 2 hours.

Liquefaction (Labomat)

Each liquefaction received ground corn (84.19% DS), backset (6.27% DS),and tap water targeting a total weight of 100 g at 32.50% Dry Solids(DS). Backset was blended at 30% w/w of total slurry weight. Initialslurry pH was approximately 5.2 and was adjusted to pH 4.8 with 40% v/vsulfuric acid prior to liquefaction. All enzymes were added according tothe experimental design listed in Table 13 below. Liquefaction tookplace in a Labomat using the following conditions: 5° C./min. Ramp, 17minute Ramp, 103 minute hold time, 40 rpm for the entire run, 200 mLstainless steel canisters. After liquefaction, all canisters were cooledin an ice bath and prepared for fermentation based on the protocollisted below under SSF.

Simultaneous Saccharification and Fermentation (SSF)

Each mash was adjusted to pH 5.0 with 50% w/w Sodium Hydroxide or 40%v/v sulfuric acid. Penicillin was applied to each mash to a totalconcentration of 3 ppm. The tubes were prepared with mash by aliquotingapproximately 4.5 g of mash per 15 mL pre-drilled test tubes to allowCO₂ release. The test tubes sat, overnight, at 4° C. until the nextmorning.

All test tubes of mash were removed from cold storage and warmed up to32° C. in the walk-in incubation chamber. Once warmed, Glucoamylase BL2,was dosed to each tube of mash at 0.50 AGU/g DS, water was added so thatall tubes received 120 μL of liquid and each mash sample received 100 μLof rehydrated yeast. Rehydrated yeast was prepared by mixing 5.5 g ofFermentis RED STAR into 100 mL of 32° C. tap water for at least 15minutes.

In monitoring CO₂ weight-loss over time, each unit of CO₂ generated andlost is converted to gram ethanol produced per gram of dry solids (gEtOH/gDS) by the following:

${g\mspace{14mu} {ethanol}\text{/}g\mspace{14mu} {DS}} = \frac{\begin{matrix}{g\mspace{14mu} {CO2}\mspace{14mu} {weight}\mspace{14mu} {loss} \times \frac{1\mspace{14mu} {mol}\mspace{14mu} {CO2}}{44.0098\mspace{14mu} g\mspace{14mu} {CO2}}} \\{\frac{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {CO2}}\frac{46.094\mspace{14mu} g\mspace{14mu} {ethanol}}{1\mspace{14mu} {mol}\mspace{14mu} {ethanol}}}\end{matrix}}{g\mspace{14mu} {mash}\mspace{14mu} {in}\mspace{14mu} {tube}\mspace{14mu} \% {DS}\mspace{14mu} {of}\mspace{14mu} {mash}}$

HPLC Analysis

Fermentation sampling took place after 54 hours of fermentation bytaking 3 tubes per treatment. Each sample was deactivated with 50 μL of40% v/v H₂SO₄, vortexing, centrifuging at 1460×g for 10 minutes, andfiltering through a 0.45 μm Whatman PP filter. 54 hour samples wereanalyzed under HPLC without further dilution. Samples were stored at 4°C. prior to and during HPLC analysis.

HPLC Agilent's 1100/1200 series with Chem station software systemDegasser, Quaternary Pump, Auto-Sampler, Column Compartment/w HeaterRefractive Index Detector (RI) Column Bio-Rad HPX- 87H Ion ExclusionColumn 300 mm × 7.8 mm part# 125-0140 Bio-Rad guard cartridge cation Hpart# 125-0129, Holder part# 125-0131 Method 0.005M H₂SO₄ mobile phaseFlow rate: 0.6 ml/min Column temperature: 65° C. RI detectortemperature: 55° C.

The method quantified analyte(s) using calibration standard for ethanol(% w/v). A four point calibration including the origin is used forquantification.

Where applicable, data were analyzed using JMP software (Cary, N.C.)with Oneway ANOVA of pairs using Tukey-Kramer HSD or Dunnett's. Errorbars denoting the 95% confidence level were established by multiplyingthe standard error of Oneway Anova analysis by 1.96.

TABLE 15 Experimental Plan. Liquefaction at 85° C. (pH 4.8) Alpha- DoseDose Dose amylase μg/g DS Protease μg/g DS Glucoamylase μg/g DS 1407 1.4— — — — 1407 1.4 Pfu 2 — — 1407 1.4 Pfu 2 PE001 10

Table 16 below shows the results:

EtOH EtOH JMP Std 95% Treatment pH (% w/v) (% Δ) Error CI Control 4.8 9.2 100% 0.022 0.042 Pfu 4.8 11.0 120% 0.022 0.042 Pfu + PE001 4.8 11.0120% 0.022 0.042

Example 10 Improved Lower Viscosity in the Ethanol Production Process

Corn Flour Preparation: Corn flour from Corn LP, Iowa, USA, was sievedand its particle size distribution (PSD) defined. U.S. Standard TestSieves with ASTM E-11 Specifications for number 12, 16, 20, 30, 40, and60 sieves were used. The dry-solids (DS) content of the received flourwas around 87.4%. Each experimental run was prepared to have the samePSD.Viscosity Profile Setup and Determination in Rapid Visco Analyzer: APerten RVA-4 unit was used for measuring the viscosity profile duringliquefaction. Corn slurries were prepared for liquefaction by weighingout specified amounts of sieved corn flour into a Perten metal cup thatreplicated the PSD of the received flour. A 40 gram slurry was made to32% DS by adding tap water and the pH-adjusted to either 5.80 (Control)using 50% w/w NaOH or 4.80 using 40% v/v H₂SO₄. Aliquots of enzyme stocksolutions were added prior to each run in the Perten RVA-4 and theamounts were also considered for getting the desired solids. The controlslurry used Alpha-Amylase A at pH 5.8. The Perten RVA-4 was programmedto mix the slurry for 1 minute at 25° C., increase the slurrytemperature from 25° C. to 85° C. in 6 minutes, hold the temperature at85° C. constant for 2 hours, cool the liquefied mash temperature from85° C. down to 32° C. in 7 minutes, and maintain the liquefied mashtemperature at 32° C. for 5 minutes. During each run, the mixing wasmaintained constant at 210 rpm.

Results

An overview of the results is provided in Table 17 and shown in FIGS.2-5.Table 17: The doses of enzymes are listed in parentheses for each andare expressed as micro g EP/g DS.

% Reduction of % % Average Reduction Reduction Viscosity of of PeakPeak- Final Average Viscosity to-Final Viscosity Exper- Viscosity vs.vs. vs. iment Enzyme Peak Peak-to- Final Experiment ExperimentExperiment No. Description Viscosity Final Viscosity 2 2 2 1Alpha-Amylase A 12769 535 1078 (1.4) at pH = 5.8 2 Alpha-Amylase 15050659 816 1407 (1.4) + Glucoamylase PE001 (10) + Protease 196 (1) at pH =4.8 3 Alpha-Amylase A 11848 728 1831 (1.4) + Glucoamylase PE001 (10) +Protease 196 (1) at pH = 4.8 4 Alpha-Amylase A 12927 527 689 14% 20% 16%(0.35) + Alpha- Amylase 1407 (1.4) + Glucoamylase PE001 (10) + Protease196 (1) at pH = 4.8 5 Alpha-Amylase A 11454 423 682 24% 36% 16% (0.7) +Alpha- Amylase 1407 (1.4) + Glucoamylase PE001 (10) + Protease 196 (1)at pH = 4.8

The present invention is further described in the following numberedparagraphs:

[1]. A process for producing fermentation products fromstarch-containing material comprising the steps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   optionally a protease having a thermostability value of more        than 20% determined as Relative Activity at 80° C./70° C.;

ii) saccharifying using a carbohydrate-source generating enzyme;

iii) fermenting using a fermenting organism.

[2]. The process of paragraph [1], further comprises, prior to theliquefaction step i), the steps of:

a) reducing the particle size of the starch-containing material,preferably by dry milling;

b) forming a slurry comprising the starch-containing material and water.

[3]. The process of paragraph [1] or [2], wherein at least 50%,preferably at least 70%, more preferably at least 80%, especially atleast 90% of the starch-containing material fit through a sieve with #6screen.[4]. The process of any of paragraphs [1]-[3], wherein the pH duringliquefaction is between 4.5-4.8.[5]. The process of any of paragraphs [1]-[4], wherein the temperatureduring liquefaction is in the range from 82−88° C., preferably around85° C.[6]. The process of any of paragraphs [1]-[5], wherein a jet-cookingstep is carried out after liquefaction in step i).[7]. The process of paragraph [6], wherein the jet-cooking is carriedout at a temperature between 110-145° C., preferably 120-140° C., suchas 125-135° C., preferably around 130° C. for about 1-15 minutes,preferably for about 3-10 minutes, especially around about 5 minutes.[8]. The process of any of paragraphs [1]-[7], wherein saccharificationand fermentation is carried out sequentially or simultaneously.[9]. The process of any of paragraphs [1]-[8], wherein saccharificationis carried out at a temperature from 20-75° C., preferably from 40-70°C., such as around 60° C., and at a pH between 4 and 5, such as about pH4.5 or about 4.8.[10]. The process of any of paragraphs [1]-[9], wherein fermentation orsimultaneous saccharification and fermentation (SSF) is carried outcarried out at a temperature from 25° C. to 40° C., such as from 28° C.to 35° C., such as from 30° C. to 34° C., preferably around about 32° C.In an embodiment fermentation is ongoing for 6 to 120 hours, inparticular 24 to 96 hours.[11]. The process of any of paragraphs [1]-[10], wherein thefermentation product is recovered after fermentation, such as bydistillation.[12]. The process of any of paragraphs [1]-[11], wherein thefermentation product is an alcohol, preferably ethanol, especially fuelethanol, potable ethanol and/or industrial ethanol.[13]. The process of any of paragraphs [1]-[12], wherein thestarch-containing starting material is whole grains.[14]. The process of any of paragraphs [1]-[13], wherein thestarch-containing material is derived from corn, wheat, barley, rye,milo, sago, cassava, manioc, tapioca, sorghum, rice or potatoes.[15]. The process of any of paragraphs [1]-[14], wherein the fermentingorganism is yeast, preferably a strain of Saccharomyces, especially astrain of Saccharomyces cerevisae.[16]. The process of any of paragraphs [1]-[15], wherein thealpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of atleast 10, such as at least 15, such as at least 20, such as at least 25,such as at least 30, such as at least 40, such as at least 50, such asat least 60, such as between 10-70, such as between 15-70, such asbetween 20-70, such as between 25-70, such as between 30-70, such asbetween 40-70, such as between 50-70, such as between 60-70.[17]. The process of any of paragraphs [1]-[16], wherein thealpha-amylase is a bacterial alpha-amylase.[18]. The process of paragraph [17], wherein the bacterial alpha-amylaseis derived from the genus Bacillus, such as a strain of Bacillusstearothermophilus, in particular a variant of a Bacillusstearothermophilus alpha-amylase, such as the one shown in SEQ ID NO: 3in WO 99/019467 or SEQ ID NO: 1 herein, in particular the Bacillusstearothermophilus alpha-amylase is truncated, preferably to have around491 amino acids.[19]. The process of any of paragraphs [1]-[18], wherein thealpha-amylase variant has at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature part of the polypeptide of SEQ ID NO: 3disclosed in WO 99/019467 or SEQ ID NO: 1 herein.[20]. The process of any of paragraphs [1]-[19], wherein thealpha-amylase is derived from Bacillus stearothermophilus alpha-amylasetruncated to have around 491 amino acids with the mutations selectedfrom the group consisting of:

-   -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+E129V+K177L+R179E; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S.        [21]. The process of any of paragraphs [1]-[20], wherein a        second alpha-amylase is added during liquefaction step i).        [22]. The process of paragraph [21], wherein the second        alpha-amylase is of bacterial origin.        [23]. The process of paragraph [21] or [22], wherein the second        alpha-amylase is derived from a strain of the genus Bacillus,        such as a strain of Bacillus stearothermophilus, in particular a        variant of a Bacillus stearothermophilus alpha-amylase, such as        the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1        herein, in particular wherein the second alpha-amylase is a        truncated Bacillus stearothermophilus alpha-amylase, preferably        to have around 491 amino acids.        [24]. The process of any of paragraphs [1]-[23], wherein the        second alpha-amylase has at least 80%, more preferably at least        85%, more preferably at least 90%, more preferably at least 91%,        more preferably at least 92%, even more preferably at least 93%,        most preferably at least 94%, and even most preferably at least        95%, such as even at least 96%, at least 97%, at least 98%, at        least 99%, or at least 100% identity to the mature part of the        polypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID        NO: 1 herein.        [25]. The process of any of paragraphs [21]-[24], wherein the        second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of below 10.        [26]. The process of any of paragraphs [21]-[25], wherein the        second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of below 8, such as below 7, such as below 6, such as        below 5.        [27]. The process of any of paragraphs [21]-[26], wherein the        second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) between 2 and 10, such as between 3 and 8, such as above        4 to 10, such as above 4 to 8.        [28]. The process of any of paragraphs [21]-[27], wherein the        second alpha-amylase is derived from Bacillus stearothermophilus        and has the following mutations I181*+G182* or I181*+G182*+N193F        (using SEQ ID NO: 1 for numbering).        [29]. The process of any of paragraphs [1]-[28], comprising the        steps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10, in particular an alpha-amylase of any one        paragraphs 16-20 and further a second alpha-amylase having a T½        (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of less than 10, in        particular a second alpha-amylase of any one of paragraphs        21-28;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C.;

ii) saccharifying using a glucoamylase;

iii) fermenting using a fermenting organism.

[30]. The process of any of paragraphs [1]-[29], wherein the proteasehas a thermostability of more than 30%, more than 40%, more than 50%,more than 60%, more than 70%, more than 80%, more than 90% more than100%, such as more that 105%, such as more than 110%, such as more than115%, such as more than 120% determined as Relative Activity at 80°C./70° C.[31]. The process of any of paragraphs [1]-[30], wherein the proteasehas a thermostability between 50 and 115%, such as between 50 and 70%,such as between 50 and 60%, such as between 100 and 120%, such asbetween 105 and 115% determined as Relative Activity at 80° C./70° C.[32]. The process of any of paragraphs [1]-[31], wherein the proteasehas a thermostability of more than 12%, more than 14%, more than 16%,more than 18%, more than 20%, more than 25%, more than 30%, more than40%, more that 50%, more than 60%, more than 70%, more than 80%, morethan 90%, more than 100%, more than 110% determined as Relative Activityat 85° C./70° C.[33]. The process of any of paragraphs [1]-[32], which protease varianthas a thermostability between 10 and 50%, such as between 10 and 30%,such as between 10 and 25% determined as Relative Activity at 85° C./70°C.[34]. The process of any of paragraphs [1]-[33], wherein thethermostability of the protease is between 50 and 110%, such as between70 and 110%, such as between 90 and 110% determined as Relative Activityat 85° C./70° C.[35]. The process of any of paragraphs [1]-[34], wherein the protease isfungal organism.[36]. The process of any of paragraph [1]-[35], wherein the protease isa variant of the metallo protease derived from a strain of the genusThermoascus, preferably a strain of Thermoascus aurantiacus, especiallyThermoascus aurantiacus CGMCC No. 0670.[37]. The process of any of paragraphs [1]-[36], wherein the protease isa variant of the metallo protease disclosed as the mature part of SEQ IDNO. 2 disclosed in WO 2003/048353 or the mature part of SEQ ID NO: 1 inWO 2010/008841 or SEQ ID NO: 3 herein.[38]. The process of any of paragraphs [1]-[37], wherein the proteasevariant has at least 75% identity preferably at least 80%, morepreferably at least 85%, more preferably at least 90%, more preferablyat least 91%, more preferably at least 92%, even more preferably atleast 93%, most preferably at least 94%, and even most preferably atleast 95%, such as even at least 96%, at least 97%, at least 98%, atleast 99%, but less than 100% identity to the mature part of thepolypeptide of SEQ ID NO: 2 disclosed in WO 2003/048353 or the maturepart of SEQ ID NO: 1 in WO 2010/008841 or SEQ ID NO: 3 herein.[39]. The process of any of paragraphs [1]-[38], wherein the protease isa variant of the Thermoascus aurantiacus protease shown in SEQ ID NO: 3with the mutations selected from the group consisting of:

-   -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L.    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L;    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L; and        [40]. The process of any of paragraphs [1]-[39], wherein the        protease is of bacterial origin.        [41]. The process of any of paragraphs [1]-[40], wherein the        protease is derived from a strain of Pyrococcus, preferably a        strain of Pyrococcus furiosus.        [42]. The process of any of paragraphs [1]-[41] wherein the        protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.        6,258,726 or SEQ ID NO: 13 herein.        [43]. The process of any of paragraphs [1]-[42], wherein the        protease is one having at least 80%, such as at least 85%, such        as at least 90%, such as at least 95%, such as at least 96%,        such as at least 97%, such as at least 98%, such as at least 99%        identity to SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or SEQ ID        NO: 13 herein.        [44]. The process of any of paragraphs [1]-[43], further wherein        a carbohydrate-source generating enzyme is present and/or added        during liquefaction step i).        [45]. The process of paragraph [44], wherein the        carbohydrate-source generating enzyme present and/or added        during liquefaction step i) is a glucoamylase.        [46]. The process of paragraph [44] or [45], wherein the        carbohydrate-source generating enzyme is a glucoamylase having a        heat stability at 85° C., pH 5.3, of at least 30%, preferably at        least 35%.        [47]. The process of any of paragraphs [44]-[46], wherein the        carbohydrate-generating enzyme is a glucoamylase having a        relative activity at pH 4.5 of at least 80%, preferably at least        85%, preferably at least 90%.        [48]. The process of any of paragraphs [44]-[47], wherein the        carbohydrate-generating enzyme is a glucoamylase having a pH        stability at pH 4.5 of at least at least 80%, at least 85%, at        least 90%, at least 95%, at least 100%.        [49]. The process of any of paragraphs [44]-[48], wherein the        carbohydrate-source generating enzyme is a glucoamylase,        preferably derived from a strain of the genus Penicillium,        especially a strain of Penicillium oxalicum disclosed as SEQ ID        NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID        NO: 9 or 14 herein.        [50]. The process of any of paragraphs [44]-[49], wherein the        glucoamylase has at least 80%, more preferably at least 85%,        more preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99% or 100% identity to the mature polypeptide shown in SEQ ID        NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID        NO: 9 or 14 herein, or wherein the carbohydrate-source        generating enzyme is a variant of the glucoamylase derived from        a strain of Penicillium oxalicum having a K79V substitution in        SEQ ID NO: 9 or 14 (using the mature sequence shown in SEQ ID        NO: 14 for numbering).        [51]. The process of any of paragraphs [1]-[50], further wherein        a glucoamylase is present and/or added during saccharification        and/or fermentation.        [52]. The process of any of paragraphs [1]-[51], wherein the        glucoamylase present and/or added during saccharification and/or        fermentation is of fungal origin, preferably from a strain of        Aspergillus, preferably A. niger, A. awamori, or A. oryzae; or a        strain of Trichoderma, preferably T. reesei; or a strain of        Talaromyces, preferably T. emersonii, or a strain of Pycnoporus,        or a strain of Gloephyllum.        [53]. The process of any of paragraphs [1]-[52], further wherein        a pullulanase is present during liquefaction and/or        saccharification.        [54]. The process of paragraph [53], wherein the pullulanase        present or added during liquefaction step i) is a family GH57        pullulanase, wherein the pullulanase preferably includes an X47        domain as disclosed in U.S. 61/289,040 published as WO        2011/087836 or shown in SEQ ID NO: 12 herein.        [55]. The process of paragraph [53] or [54], wherein the        pullulanase is derived from a strain from the genus        Thermococcus, including Thermococcus litoralis and Thermococcus        hydrothermalis or a hybrid thereof.        [56]. The process of any of paragraphs [53]-[55], wherein the        pullulanase is the truncated Thermococcus hydrothermalis        pullulanase at site X4 or a T. hydrothermalis/T. litoralis        hybrid enzyme with truncation site X4 disclosed in U.S.        61/289,040 published as WO 2011/087836 or shown in SEQ ID NO: 12        herein.        [57]. The process of any of paragraphs [1]-[56], comprising the        steps of:

i) liquefying the starch-containing material at a pH in the range from4.5-5.0 at a temperature in the range from 80-90° C. using:

-   -   an alpha-amylase derived from Bacillus stearothermophilus having        a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of at least 10;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C. derived from        Pyrococcus furiosus or Thermoascus aurantiacus;

ii) saccharifying using a glucoamylase enzyme;

iii) fermenting using a fermenting organism.

[58]. A composition comprising an alpha-amylase and a protease, whereinthe

i) alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂) of atleast 10;

ii) protease having a thermostability value of more than 20% determinedas Relative Activity at 80° C./70° C.

[59]. The composition of paragraph [58], further comprising acarbohydrate-source generating enzyme.[60]. The composition of paragraph [58] or [59], wherein thecarbohydrate-source generating enzyme is a glucoamylase having aRelative Activity heat stability at 85° C. of at least 20%, at least30%, preferably at least 35%.[61]. The composition of any of paragraphs [58]-[60], wherein thealpha-amylase is a bacterial alpha-amylase, in particular of the genusBacillus, such as a strain of Bacillus stearothermophilus, in particulara variant of a Bacillus stearothermophilus alpha-amylase, such as theone shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1 herein, inparticular wherein the Bacillus stearothermophilus alpha-amylase variantis truncated, preferably to have around 491 amino acids.[62]. The process of any of paragraphs [58]-[61], wherein thealpha-amylase variant has at least 80%, more preferably at least 85%,more preferably at least 90%, more preferably at least 91%, morepreferably at least 92%, even more preferably at least 93%, mostpreferably at least 94%, and even most preferably at least 95%, such aseven at least 96%, at least 97%, at least 98%, at least 99%, but lessthan 100% identity to the mature part of the polypeptide of SEQ ID NO: 3disclosed in WO 99/019467 or SEQ ID NO: 1 herein.[63]. The composition of any of paragraphs [58]-[62], wherein thealpha-amylase is derived from Bacillus stearothermophilus alpha-amylasetruncated to have around 491 amino acids with the mutations selectedfrom the group consisting of:

-   -   I181*+G182*+N193F+V59A+Q89R+E129V+K177L+R179E+H208Y+K220P+N224L+Q254S;    -   I181*+G182*+N193F+E129V+K177L+R179E; and    -   I181*+G182*+N193F+E129V+K177L+R179E+K220P+N224L+S242Q+Q254S.        [64]. The composition of any of paragraphs [58]-[63], wherein        the alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 15, such as at least 20, such as at least 25,        such as at least 30, such as at least 40, such as at least 50,        such as at least 60, such as between 10-70, such as between        15-70, such as between 20-70, such as between 25-70, such as        between 30-70, such as between 40-70, such as between 50-70,        such as between 60-70.        [65]. The composition of any of paragraphs [58]-[64], further        wherein the composition comprises a second alpha-amylase, in        particular of bacterial origin.        [66]. The composition of paragraph [65], wherein the second        alpha-amylase is derived from a strain of the genus Bacillus,        such as a strain of Bacillus stearothermophilus, in particular a        variant of a Bacillus stearothermophilus alpha-amylase, such as        the one shown in SEQ ID NO: 3 in WO 99/019467 or SEQ ID NO: 1        herein, in particular wherein the second alpha-amylase is a        truncated Bacillus stearothermophilus alpha-amylase, preferably        to have around 491 amino acids.        [67]. The process of any of paragraphs [58]-[66], wherein the        second alpha-amylase has at least 80%, more preferably at least        85%, more preferably at least 90%, more preferably at least 91%,        more preferably at least 92%, even more preferably at least 93%,        most preferably at least 94%, and even most preferably at least        95%, such as even at least 96%, at least 97%, at least 98%, at        least 99%, or at least 100% identity to the mature part of the        polypeptide of SEQ ID NO: 3 disclosed in WO 99/019467 or SEQ ID        NO: 1 herein.        [68]. The composition of any of paragraphs [65]-[67], wherein        the second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12        mM CaCl₂) of below 10.        [69]. The composition of any of paragraphs [65]-[68], wherein        the second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12        mM CaCl₂) of below 8, such as below 7, such as below 6, such as        below 5.        [70]. The composition of any of paragraphs [65]-[69], wherein        the second alpha-amylase has a T½ (min) at pH 4.5, 85° C., 0.12        mM CaCl₂) between 2 and 10, such as between 3 and 8, such as        above 4 to 10, such as above 4 to 8.        [71]. The composition of paragraph [64]-[70], wherein the second        alpha-amylase is derived from Bacillus stearothermophilus and        has the following mutations I181*+G182* or I181*+G182*+N193F        (using SEQ ID NO: 1 for numbering).        [72]. The composition of any of paragraphs [58]-[71],        comprising:    -   an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM        CaCl₂) of at least 10;    -   a second alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12        mM CaCl₂) of less than 10;    -   a protease having a thermostability value of more than 20%        determined as Relative Activity at 80° C./70° C.;    -   a thermostable glucoamylase.        [73]. The composition of paragraph [72], wherein the        thermostable alpha-amylase is one of any of paragraphs 61-64.        [74]. The composition of paragraph [72] or [73], wherein the        second alpha-amylase is one of any of paragraphs 65-71.        [75]. The composition of any of paragraphs [58]-[74], wherein        the protease has a thermostability of more than 30%, more than        40%, more than 50%, more than 60%, more than 70%, more than 80%,        more than 90% more than 100%, such as more that 105%, such as        more than 110%, such as more than 115%, such as more than 120%        determined as Relative Activity at 80° C./70° C.        [76]. The composition of any of paragraphs [58]-[75], wherein        the protease has a thermostability between 50 and 115%, such as        between 50 and 70%, such as between 50 and 60%, such as between        100 and 120%, such as between 105 and 115% determined as        Relative Activity at 80° C./70° C.        [77]. The composition of any of paragraphs [58]-[76], wherein        the protease has a thermostability of more than 12%, more than        14%, more than 16%, more than 18%, more than 20%, more than 25%,        more than 30%, more than 40%, more that 50%, more than 60%, more        than 70%, more than 80%, more than 90%, more than 100%, more        than 110% determined as Relative Activity at 85° C./70° C.        [78]. The composition of any of paragraphs [58]-[77], wherein        the protease has a thermostability of between 10 and 50%, such        as between 10 and 30%, such as between 10 and 25% determined as        Relative Activity at 85° C./70° C.        [79]. The composition of any of paragraphs [58]-[78], wherein        the protease has a thermostability between 50 and 110%, such as        between 70 and 110%, such as between 90 and 110% determined as        Relative Activity at 85° C./70° C.        [80]. The composition of any of paragraphs [58]-[79], wherein        the protease is a variant of the metallo protease derived from        Thermoascus aurantiacus CGMCC No. 0670 show in SEQ ID NO: 3        herein.        [81]. The composition of any of paragraphs [58]-[80], wherein        the protease variant of the Thermoascus aurantiacus protease        shown in SEQ ID NO: 3 with the mutations selected from the group        consisting of:    -   A27K+D79L+Y82F+S87G+D104P+A112P+A126V+D142L;    -   D79L+Y82F+S87G+A112P+D142L;    -   Y82F+S87G+S70V+D79L+D104P+A112P+D142L; and    -   Y82F+S87G+D79L+D104P+A112P+A126V+D142L.        [82]. The composition of any of paragraphs [58]-[81], wherein        the protease is derived from a strain of Pyrococcus, preferably        a strain of Pyrococcus furiosus.        [83]. The composition of any of paragraphs [58]-[82], wherein        the protease is the one shown in SEQ ID NO: 1 in U.S. Pat. No.        6,258,726 or SEQ ID NO: 13 herein.        [84]. The composition of any of paragraphs [58]-[83], wherein        the protease is one having at least 80%, such as at least 85%,        such as at least 90%, such as at least 95%, such as at least        96%, such as at least 97%, such as at least 98%, such as at        least 99% identity to SEQ ID NO: 1 in U.S. Pat. No. 6,258,726 or        SEQ ID NO: 13 herein.        [85]. The composition of any of paragraphs [58]-[84], further        comprising a carbohydrate-source generating enzyme, in        particular a glucoamylase, which has a heat stability at 85° C.,        pH 5.3, of at least 30%, preferably at least 35%.        [86]. The composition of paragraph [85], wherein the        carbohydrate-generating enzyme is a glucoamylase having a        relative activity at pH 4.5 of at least 80%, preferably at least        85%, preferably at least 90%.        [87]. The composition of paragraph [85] or [86], wherein        carbohydrate-generating enzyme is a glucoamylase having a pH        stability at pH 4.5 of at least at least 80%, at least 85%, at        least 90%, at least 95%, at least 100%.        [88]. The composition of any of paragraphs [85]-[87], wherein        the carbohydrate-source generating enzyme is a glucoamylase,        preferably derived from a strain of the genus Penicillium,        especially a strain of Penicillium oxalicum disclosed as SEQ ID        NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or SEQ ID        NO: 9 or 14 herein.        [89]. The composition of any of paragraphs [85]-[88], wherein        the carbohydrate-source generating enzyme is a variant of the        glucoamylase derived from a strain of Penicillium oxalicum        disclosed as SEQ ID NO: 2 in WO 2011/127802 having a K79V        substitution in SEQ ID NO: 9 or 14 (using the mature sequence        shown in SEQ ID NO: 14 for numbering).        [90]. The composition of any of paragraphs [85]-[89], wherein        the glucoamylase has at least 80%, more preferably at least 85%,        more preferably at least 90%, more preferably at least 91%, more        preferably at least 92%, even more preferably at least 93%, most        preferably at least 94%, and even most preferably at least 95%,        such as even at least 96%, at least 97%, at least 98%, at least        99% or 100% identity to the mature polypeptide shown in SEQ ID        NO: 2 in PCT/CN10/071753 published as WO 2011/127802 or shown in        SEQ ID NO: 9 or 14 herein.        [91]. The composition of paragraphs [58]-[90], further        comprising a pullulanase.        [92]. The composition of paragraph [91], wherein the pullulanase        is a GH57 pullulanase, which preferably includes an X47 domain        as disclosed in U.S. 61/289,040 published as WO 2011/087836.        [93]. The composition of paragraph [91] or [92], wherein the        pullulanase is derived from a strain from the genus        Thermococcus, including Thermococcus litoralis and Thermococcus        hydrothermalis shown in SEQ ID NO: 10 herein, or a hybrid        thereof.        [94]. The composition of paragraph [93], wherein the pullulanase        is the truncated Thermococcus hydrothermalis pullulanase at site        X4 or a T. hydrothermalis/T. litoralis hybrid enzyme with        truncation site X4 disclosed in U.S. 61/289,040 published as WO        2011/087836 or shown in SEQ ID NO: 12 herein.        [95]. The composition of any of paragraphs [58]-[94] comprising:

i) an alpha-amylase having a T½ (min) at pH 4.5, 85° C., 0.12 mM CaCl₂)of at least 10 derived from Bacillus stearothermophilus;

ii) a protease having a thermostability value of more than 20%determined as Relative Activity at 80° C./70° C. derived derived fromPyrococcus furiosus or Thermoascus aurantiacus; and optionally

iii) a glucoamylase derived from Penicillium oxalicum.

[96]. The composition of paragraph [95], wherein the composition furthercomprising a second alpha-amylase having a T½ (min) at pH 4.5, 85° C.,0.12 mM CaCl₂) of less than 10 derived from Bacillus stearothermophilus.[97]. Use of a composition of any of paragraphs [58]-[96] in aliquefaction process.[98]. The use according to paragraph 97, wherein liquefaction is carriedout as defined in any of paragraphs [1]-[57].

1. A process for producing a fermentation product from astarch-containing material comprising: i) liquefying thestarch-containing material at a temperature from 80°-90° C. using: analpha-amylase and a thermostable protease having a thermostability valueof more than 80% determined as Relative Activity at 80° C./70° C.; ii)saccharifying the liquefied material obtained in step i) using acarbohydrate-source generating enzyme; and iii) fermenting thesaccharified material obtained in step (ii) using a fermenting organism.2. The process of claim 1, wherein the alpha-amylase has a T½ (min) atpH 4.5, 85° C., 0.12 mM CaCl2) of at least
 30. 3. The process of claim1, wherein the alpha-amylase is a variant of a Bacillus alpha-amylase.4. The process of claim 1, wherein the protease is a serine protease. 5.The process of claim 1, wherein the protease is from a strain ofPyrococcus.
 6. The process of claim 1, wherein a thermostableglucoamylase is present and/or added during liquefaction step i).
 7. Theprocess of claim 6, wherein the glucoamylase has a heat stability at 85°C., pH 5.3, of at least 30%.
 8. The process of claim 6, wherein theglucoamylase is from a strain of the genus Penicillium.
 9. The processof claim 1, wherein the carbohydrate-source generating enzyme is aglucoamylase.
 10. The process of claim 1, wherein the fermentationproduct comprises fuel ethanol.
 11. The process of claim 1, wherein thestarch-containing material comprises corn.
 12. The process of claim 1,wherein steps (ii) and (iii) are performed simultaneously.
 13. Theprocess of claim 1, wherein the fermenting organism is yeast.
 14. Theprocess of claim 14, wherein the yeast is a strain of Saccharomyces. 15.The process of claim 1, wherein the pH during liquefying step (i) isfrom 4.5-5.0.
 16. A process for producing a fermentation product from astarch-containing material comprising: i) liquefying thestarch-containing material at a temperature from 80°-90° C. using: analpha-amylase and a thermostable protease having a thermostability valueof more than 90% determined as Relative Activity at 80° C./70° C.; ii)saccharifying the liquefied material obtained in step i) using acarbohydrate-source generating enzyme; and iii) fermenting thesaccharified material obtained in step (ii) using a fermenting organism.17. The process of claim 16, wherein the alpha-amylase has a T½ (min) atpH 4.5, 85° C., 0.12 mM CaCl2) of at least
 30. 18. The process of claim16, wherein the alpha-amylase is a variant of a Bacillus alpha-amylase.19. The process of claim 16, wherein the protease is a serine protease.20. The process of claim 16, wherein the protease is from a strain ofPyrococcus.
 21. The process of claim 16, wherein a thermostableglucoamylase is present and/or added during liquefaction step i). 22.The process of claim 21, wherein the glucoamylase has a heat stabilityat 85° C., pH 5.3, of at least 30%.
 23. The process of claim 21, whereinthe glucoamylase is from a strain of the genus Penicillium.
 24. Theprocess of claim 16, wherein the carbohydrate-source generating enzymeis a glucoamylase.
 25. The process of claim 16, wherein the fermentationproduct comprises fuel ethanol.
 26. The process of claim 16, wherein thestarch-containing material comprises corn.
 27. The process of claim 16,wherein steps (ii) and (iii) are performed simultaneously.
 28. Theprocess of claim 16, wherein the fermenting organism is yeast.
 29. Theprocess of claim 28, wherein the yeast is a strain of Saccharomyces. 30.The process of claim 16, wherein the pH during liquefying step i) isfrom 4.5-5.0.
 31. A process for producing a fermentation product from astarch-containing material comprising: i) liquefying thestarch-containing material at a temperature from 80°-90° C. using: analpha-amylase and a thermostable protease having a thermostability valueof more than 100% determined as Relative Activity at 80° C./70° C.; ii)saccharifying the liquefied material obtained in step i) using acarbohydrate-source generating enzyme; and iii) fermenting thesaccharified material obtained in step (ii) using a fermenting organism.32. The process of claim 31, wherein the alpha-amylase has a T½ (min) atpH 4.5, 85° C., 0.12 mM CaCl2) of at least
 30. 33. The process of claim31, wherein the alpha-amylase is a variant of a Bacillus alpha-amylase.34. The process of claim 31, wherein the protease is a serine protease.35. The process of claim 31, wherein the protease is from a strain ofPyrococcus.
 36. The process of claim 31, wherein a thermostableglucoamylase is present and/or added during liquefaction step i). 37.The process of claim 36, wherein the glucoamylase has a heat stabilityat 85° C., pH 5.3, of at least 30%.
 38. The process of claim 36, whereinthe glucoamylase is from a strain of the genus Penicillium.
 39. Theprocess of claim 31, wherein the carbohydrate-source generating enzymeis a glucoamylase.
 40. The process of claim 31, wherein the fermentationproduct comprises fuel ethanol.
 41. The process of claim 31, wherein thestarch-containing material comprises corn.
 42. The process of claim 31,wherein steps (ii) and (iii) are performed simultaneously.
 43. Theprocess of claim 31, wherein the fermenting organism is yeast.
 44. Theprocess of claim 43, wherein the yeast is a strain of Saccharomyces. 45.The process of claim 31, wherein the pH during liquefying step i) isfrom 4.5-5.0.